NZ711775B2

NZ711775B2 - Genetic modification of rats

Info

Publication number: NZ711775B2
Application number: NZ711775A
Authority: NZ
Inventors: Wojtek Auerbach; David Frendewey; David Heslin; Ka Man Venus Lai; Jeffrey D Lee; David M Valenzuela
Original assignee: Regeneron Pharmaceuticals Inc
Priority date: 2013-02-20
Filing date: 2014-02-20
Publication date: 2021-05-27

Abstract

Compositions and methods are provided for making rat pluripotent and totipotent cells, including rat embryonic stem (ES) cells. Compositions and methods for improving efficiency or frequency of germline transmission of genetic modifications in rats are provided. Such methods and compositions comprise an in vitro culture comprising a feeder cell layer and a population of rat ES cells or a rat ES cell line, wherein the in vitro culture conditions maintain pluripotency of the ES cell and comprises a media having mouse leukemia inhibitor factor (LIF) or an active variant or fragment thereof. Various methods of establishing such rat ES cell lines are further provided. Methods of selecting genetically modified rat ES cells are also provided, along with various methods to generate a transgenic rat from the genetically modified rat ES cells provided herein. Various kits and articles of manufacture are further provided. e an in vitro culture comprising a feeder cell layer and a population of rat ES cells or a rat ES cell line, wherein the in vitro culture conditions maintain pluripotency of the ES cell and comprises a media having mouse leukemia inhibitor factor (LIF) or an active variant or fragment thereof. Various methods of establishing such rat ES cell lines are further provided. Methods of selecting genetically modified rat ES cells are also provided, along with various methods to generate a transgenic rat from the genetically modified rat ES cells provided herein. Various kits and articles of manufacture are further provided.

Description

GENETIC MODIFICATION OF RATS FIELD Non-human pluripotent, totipotent, and embryonic stem (ES) cells, in particular rat otent, totipotent, and/or rat ES cells, and methods of making them. Methods for making rat pluripotent, totipotent, and ES cells are provided. Methods for targeting rat pluripotent, totipotent, and/or ES cells are provided. Methods for achieVing germline transmission of a genetic modiﬁcation in a rat cell are provided. Media for deriving, growing, and maintaining rat otent, totipotent, and ES cells are provided.

NCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB The ofﬁcial copy of the sequence listing is submitted concurrently with the cation as a text ﬁle Via EFS-Web, in compliance with the American Standard Code for Information Interchange (ASCII), with a ﬁle name of seqlist.txt, a creation date of ry 20, 2014 and a size of 2 Kb. The sequence listing ﬁled Via EFS-Web is part of the speciﬁcation and is hereby incorporated in its entirety by reference herein.

OUND The rat has been a valuable model for many applications, including, but not limited to, applications in drug discovery. The usefulness of the rat has been mitigated somewhat by difﬁculty in obtaining genetically modiﬁed rats, in ular, in developing methods for genetically modifying rats, and generating useful rat cells that can be used in genetic modiﬁcation ols, including but not limited to protocols that result in germline transmission of a genetic modiﬁcation in a rat genome.

There is a need in the art for rat cells (e. g., embryonic stem cells) that can be genetically modiﬁed such that the genetic modiﬁcation can be transmitted h the germline. There is a need in the art for improved frequency of germline ission of genetic modiﬁcations in rats.

There is a need in the art for donor rat pluripotent, totipotent, and/or ES cells from various strains of rat that are capable of generating F0, or wholly donor cell- derived, F0 rats. There is a need in the art for donor rat pluripotent, totipotent, and/or ES cells that are capable of generating rats that comprise a germline genetic modiﬁcation.

SUMMARY Compositions and methods are provided for making rat pluripotent and/or totipotent cells, including rat embryonic stem (ES) cells. Compositions and methods for improving ncy or frequency of germline transmission of genetic modiﬁcations in rats are provided. In various s, the s and compositions comprise an in vitro culture comprising a feeder cell layer and a tion of rat ES cells or a rat ES cell line, wherein the in vitro culture ions allow nance of pluripotency of the rat ES cell. Various methods of ishing rat ES cell lines are further provided. Method of selecting genetically modiﬁed rat ES cells are also provided, along with various methods to generate a transgenic rat from the genetically modiﬁed rat ES cells are ed herein. Various kits and articles of manufacture are further provided.

Non-limiting embodiments are as follows: 1. An ed rat ES cell of a strain selected from ACI or DA, wherein the isolated rat ES cell is and capable of transmitting its genome through the germline. 2. The isolated rat ES cell of embodiment 1, wherein the cell is derived from an ACI rat. 3. The isolated rat ES cell of embodiment l or 2, wherein the cell is derived from a Dark Agouti (DA) rat.2. 4. The isolated rat ES cell of embodiment l, 2, or 3, wherein the cell is euploid and capable of transmitting a ed genetic modiﬁcation through the ne.

. The isolated rat ES cell of embodiment 4, wherein the rat ES cell comprises a germline transmission efﬁciency of the targeted genetic modiﬁcation of at least 3%. 6. The isolated rat ES cell of embodiment 4, wherein the rat ES cell has a germline transmission efﬁciency of the targeted genetic ation of at least 60%. 7. The isolated rat ES cell of any one of embodiments 1-6, wherein the rat ES cell exhibits a targeting efﬁciency of homologous recombination of at least 2%. 8. The isolated rat ES cell of any one of embodiments 1-8, wherein the rat ES cell is capable of transmitting a targeted genetic modiﬁcation into progeny ing a successive round of electroporation. 9. The isolated rat ES cells of any one of embodiments 1-8, wherein the rat ES cell comprises one or more, two or more, or three or more targeted genetic modiﬁcation.

. The isolated rat ES cell of any one of embodiments 4-9, wherein the targeted genetic modiﬁcation comprises an insertion, a deletion, a ut, a knockin, a point mutation, or a ation thereof. 11. The isolated rat ES cell of embodiment 9, wherein the targeted genetic ation comprises at least one insertion of a heterologous polynucleotide into a genome of the cell. 12. The ed rat ES cell of embodiment 11, wherein the heterologous polynucleotide comprises a selection marker. 13. The isolated rat ES cell of embodiment 12, wherein (a) the selection marker comprises a non-attenuated ion marker gene ly linked to a promoter; or (b) the rat ES cell comprises at least 2 copies of the polynucleotide encoding the selection marker. 14. The isolated rat ES cell of embodiment 12, wherein the selection marker has an sed actiVity compared to a wild type selection marker. 15. The isolated rat ES cell of any one of embodiments 1-14, wherein the rat ES cell forms a sphere-like colony when plated on a feeder cell layer in culture comprising a LIF polypeptide, a GSK3 inhibitor, and a MEK inhibitor. 16. The isolated rat ES cell of any one of ments 1-15, wherein the rat ES cell, when cultured in vitro, loosely adhere to the feeder cell layer. 17. The isolated rat ES cell of any one of embodiments 1-16, wherein the cell does not require ine LIF signaling for maintenance of pluripotency. 18. The isolated rat ES cell of any one of embodiments 1-17, wherein the cell is a male (XY) rat ES cell. 19. The isolated rat ES cell of any one of embodiments 1-19, wherein the cell is a female (XX) rat ES cell.

. The isolated rat ES cell of any one of embodiments 1-19, wherein the rat ES cell can be passaged up to at least 11 times in a medium comprising a GSK3 inhibitor and a MEK inhibitor without decreasing its ing efﬁciency or ne transmission efﬁciency of a targeted genetic modiﬁcation. 21. The isolated rat ES cell of any one of embodiments 1-20, wherein the rat ES cells express at least one pluripotency marker selected from Dnmt3 L, Eras, Err-beta, FbxolS, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a combination thereof 22. The isolated rat ES cell of any one of embodiments l-2l, wherein the rat ES cells do not express one or more pluripotency markers selected from c-Myc, Ecatl, Rexol, or a combination f. 23. The isolated rat ES cell of any one of embodiments l-22, wherein the rat ES cells do not express one or more mesodermal markers ed from Brachyury, Bmpr2, or a combination thereof. 24. The ed rat ES cell of any one of embodiments l-23, wherein the rat ES cells do not express one or more endodermal markers selected from Gata6, Soxl7, Sox7, or combination thereof; 25. The isolated rat ES cell of any one of embodiments l-24, wherein the rat ES cells do not express one or more neural markers selected from Nestin, Pax6, or combination thereof 26. The isolated rat ES cell of any one of embodiments 1-25, wherein the cell expresses a pluripotency marker comprising Oct-4, Sox2, ne phosphatase, or a combination thereof 27. The isolated rat ES cell of any one of embodiments 1-26, wherein the rat ES cell is characterized by the expression of one or more of a rat ESC-speciflc gene selected from one or more of s Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine nucleotide ge factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-Al (Efnal), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gij), Insulin-like growth factor binding protein-like l (Igfbpll), Interleukin 36 beta(Illf8), Interleukin 28 receptor, alpha (Il28ra), left-right determination factor 1 (Leftyl), ia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 (Ptpnl 8), Caudal type homeobox 2 , Fibronectin type III and ankyrin repeat domains 1 (Fankl), Forkhead box El id transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box El (thyroid transcription factor 2) (Foxel), Hairy/enhancer-ofsplit related with YRPW motif 2 (Hey2), Lymphoid er-binding factor 1 (Lefl), ke 3 (Drosophila) (Sall3), SATB homeobox l (Satbl), 2, or a combination thereof 28. An isolated tion of rat ES cells, wherein at least 70% of the rat ES cells are euploid and form sphere-like colonies when plated on a feeder cell layer in vitro. 29. The isolated population of rat ES cells of embodiment 28, wherein the rat ES cells are d from an AC1 rat.

. The isolated population of rat ES cells of embodiment 28, wherein the rat ES cells are derived from a Dark Agouti (DA) rat. 31. The ed population of rat ES cells of any one of embodiments 28-30, wherein the rat ES cells are capable of transmitting their genome through the germline. 32. The isolated population of rat ES cells of any one of embodiments 28-3l, wherein the rat ES cells have a germline transmission ncy of the targeted genetic modiﬁcation of at least 3%. 33. The isolated population of rat ES cells of any one of embodiments 28-3l, wherein the rat ES cells have a germline transmission efﬁciency of the targeted genetic modiﬁcation of at least 60%. 34. The ed population of rat ES cells of any one of embodiments 28-3l, wherein the rat ES cells exhibit a targeting efﬁciency of homologous recombination of at least 2%.

. The isolated population of rat ES cells of any one of embodiments 28-34, wherein the rat ES cells are capable of transmitting a ed genetic modiﬁcation into progeny following a successive round of oporation. 36. The isolated population of any one embodiments 28-35, wherein the rat ES cells comprise one or more, two or more, or three or more targeted genetic modiﬁcation and can transmit the targeted genetic modiﬁcation through the germline. 37. The isolated population of rat ES cells of embodiment 36, wherein the targeted genetic modiﬁcation is at the rat Rosa26 locus. 38. The isolated population of rat ES cells of embodiment 36, n the targeted genetic ation comprises an insertion, a deletion, a knockout, a knockin, a point mutation, or a combination thereof. 39. The isolated population of rat ES cells of embodiment 36, wherein the targeted genetic modiﬁcation comprises at least one insertion of a heterologous polynucleotide into a genome of the cell. 40. The isolated population of rat ES cells of embodiment 39, n the logous polynucleotide comprises a selection . 41. The ed population of rat ES cells of embodiment 40, wherein (a) the selection marker comprises a non-attenuated selection marker gene operably linked to a promoter; or (b) the rat ES cell comprises at least 2 copies of the polynucleotide encoding the selection . 42. The isolated population of rat ES cells of embodiment 40, wherein the selection marker has an increased activity compared to a wild type selection marker 43. The isolated population of rat ES cells of any one of embodiments 28-42, wherein the cells form a sphere-like colony when plated on a feeder cell layer in culture comprising a LIF polypeptide, a GSK3 tor, and a MEK tor. 44. The isolated population rat ES cells of any one of embodiments 28-43, wherein the cells, when cultured in vitro, loosely adhere to the feeder cell layer. 45. The isolated population of rat ES cells of any one of embodiments 28-44, n the cells do not require paracrine LIF ing for maintenance of pluripotency. 46. The isolated population of rat ES cells of any one of embodiments 28-44, wherein the rat ES cells are a male (XY) rat ES cells. 47. The isolated population of rat ES cells of any one of embodiments 28-44, wherein the rat ES cells are female (XX) rat ES cells. 48. The isolated population of rat ES cells of any one of ments 28-47, wherein the rat ES cells can be passaged up to at least ll times in a medium comprising a GSK3 inhibitor and a MEK inhibitor without decreasing its ing efﬁciency or germline transmission efﬁciency of a targeted genetic modiﬁcation. 49. The isolated population of rat ES cells of any one of embodiments 28-48, wherein the rat ES cells express at least one pluripotency marker selected from Dnmt3 L, Eras, Err-beta, Fbxol5, Fgf4, Gdf3, Klf4, Lefl, LIF receptor, Lin28, Nanog, Oct4, Soxl5, Sox2, Utfl, or a combination thereof. 50. The isolated population of rat ES cells of any one of embodiments 28-49, wherein the rat ES cells do not s one or more pluripotency markers selected from c-Myc, Ecatl , Rexol, or a combination thereof 51. The ed population of rat ES cells of any one of embodiments 28-50, wherein the rat ES cells do not express one or more mesodermal markers selected from Brachyury, Bmpr2, or a combination thereof 52. The isolated population of rat ES cells of any one of embodiments 28-51, n the rat ES cells do not express one or more endodermal markers selected from Gata6, Soxl7, Sox7, or combination thereof; 53. The isolated population of rat ES cells of any one of embodiments 28-52, wherein the rat ES cells do not express one or more neural markers selected from Nestin, Pax6, or combination thereof. 54. The isolated population of rat ES cells of any one of embodiment 28-53, wherein the rat ES cells ses a pluripotency marker comprising Oct-4, Sox2, alkaline phosphatase, or a combination thereof. 55. The isolated population of rat ES cells of any one of embodiments 28-54, wherein the rat ES cells are characterized by the expression of one or more of a rat ESC- speciﬁc gene selected from one or more of Adheres Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9 f9), Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-Al (Efnal), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gij), Insulin-like growth factor binding n-like l (Igfbpll), Interleukin 36 beta(Illf8), Interleukin 28 receptor, alpha (Il28ra), left-right determination factor 1 (Leftyl), Leukemia inhibitory factor receptor alpha (Lifr), osphatidic acid receptor 2 ), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III and ankyrin repeat domains 1 ), Forkhead box El (thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box El (thyroid ription factor 2) ), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Lymphoid enhancer-binding factor 1 , Sal-like 3 (Drosophila) (Sall3), SATB homeobox l (Satbl), miR-632, or a ation thereof 56. The isolated population of rat ES cells of any one of embodiments 28-55 wherein the population comprises at least 104 cells. 57. The isolated population of rat ES cells of any one of embodiments 28-56, wherein the rat ES cells have one or more characteristic comprising: a. at least 90% of the rat ES cells are euploid; b. at least 70% of the rat ES cells express at least one pluripotency ; wherein the at least one pluripotency marker comprises Oct-4, Sox2, alkaline phosphatase, or a combination thereof; c. a cell from the rat ES cell population, when ed with a rat host embryo transmits the genome of the rat ES cell line into an offspring; d. the rat ES cells when cultured in vitro loosely adhere to a feeder cell layer; e. the rat ES cells form sphere-like colonies when plated on a feeder cell layer in vitro; (f) the rat ES cells maintain pluripotency when cultured in vitro in a media comprising an GSK3 inhibitor, a MEK inhibitor, LIF and a feeder cell layer that is not genetically modiﬁed to express LIF; f. the rat ES cell exhibits a targeting efﬁciency of homologous recombination of at least 2%; g. the rat ES cells maintain pluripotency in vitro without requiring ine LIF signaling; h. at least 70% of the rat ES cells are euploid and form sphere-like colonies when plated on a feeder cell layer in vitro; i. the rat ES cells express at least one otency marker selected from Dnmt3 L, Eras, Err-beta, FbxolS, Fgf4, Gdf3, Klf4, Lefl, LIF receptor, Lin28, Nanog, Oct4, SoxlS, Sox2, Utfl, or a combination thereof; j. the rat ES cells do not express one or more differentiation markers selected from c-Myc, Ecatl, Rexol; k. the rat ES cells do not express one or more mesodermal markers selected from Brachyury, Bmpr2, or a combination f; 1. the rat ES cells do not express one or more rmal s ed from Gata6, Soxl7, Sox7, or combination thereof; and/or m. the rat ES cells do not express one ore more neural markers selected from Nestin, Pax6, or combination thereof 58. The isolated tion of rat ES cells of any one of embodiments 28-57, wherein (a) the rat ES cells is derived from a rat blastocyst; (b) the rat ES cell is derived from a rat morula stage embryo; and/or, (c) the rat ES cell line is derived from a superovulated rat. 59. An in vitro culture comprising a feeder cell layer, the population of rat embryonic stem (ES) cells, and a medium sing a Leukemia Inhibitory Factor (LIF), GSK3 inhibitor, and a MEK inhibitor, n at least 70% of the rat ES cells are euploid and the rat ES cell forms a sphere-like colony. 60. The in vitro culture of embodiment 59 or 60, wherein the rat ES cell, loosely adhere to the feeder cell layer. 61. The in vitro culture of embodiment 59, 60, or 61, wherein the rat ES cells are capable of transmitting their genome through the germline. 62. The in vitro culture of embodiment 59, 60 or 61, wherein the rat ES cells are derived from an AC1 rat. 63. The in vitro culture of ment 59, 60 or 61, wherein the rat ES cells are derived from a Dark Agouti (DA) rat.2. 64. The in vitro culture of any one of embodiments 59-63, wherein the rat ES cells are capable of transmitting a targeted genetic modiﬁcation through the germline. 65. The in vitro culture of embodiment 64, wherein the rat ES cells comprise a ne transmission efﬁciency of the targeted genetic modiﬁcation of at least 3%. 66. The in vitro culture of ment 64, wherein the rat ES cells have a germline transmission efﬁciency of the targeted genetic modiﬁcation of at least 60%. 67. The in vitro culture of any one of embodiments 59-66, wherein the rat ES cells exhibit a targeting ncy of gous recombination of at least 2%. 68. The in vitro culture of any one of embodiments 59-67, wherein the rat ES cell is capable of transmitting a ed genetic modiﬁcation into progeny following a successive round of electroporation. 69. The in vitro culture of any one of embodiments 59-68, wherein the rat ES cell comprises one or more, two or more, or three or more targeted genetic modiﬁcation. 70. The in vitro e of embodiment 69, wherein the targeted genetic modiﬁcation comprises an insertion, a deletion, a knockout, a knockin, a point mutation, or a combination thereof 71. The in vitro e of embodiment 69, wherein the targeted genetic modiﬁcation comprises at least one insertion of a heterologous polynucleotide into a genome of the cell. 72. The in vitro culture of ment 71, wherein the heterologous polynucleotide comprises a selection marker. 73. The in vitro culture of embodiment 72, wherein (a) the selection marker comprises a non-attenuated ion marker gene operably linked to a promoter; or (b) the rat ES cell comprises at least 2 copies of the polynucleotide encoding the ion marker. 74. The in vitro culture of embodiment 72, n the selection marker has an increased activity compared to a wild type selection marker. 75. The in vitro culture of any one of embodiments 59-74, wherein the cell does not require paracrine LIF signaling for maintenance of pluripotency. 76. The in vitro culture of embodiment of any one of embodiments 59-75, wherein the cell is a male (XY) rat ES cell. 77. The in vitro culture of embodiment of any one of embodiments 59-75, wherein the cell is a female (XX) rat ES cell. 78. The in vitro culture of embodiment of any one of ments 59-77, n the rat ES cell can be passaged up to at least ll times in a medium comprising a GSK3 inhibitor and a MEK tor without decreasing its targeting efficiency or germline transmission efﬁciency of a targeted genetic modification. 79. The in vitro culture of any one of embodiments 59-78, wherein the rat ES cells s at least one pluripotency marker selected from Dnmt3 L, Eras, Err-beta, FbxolS, Fgf4, Gdf3, Klf4, Lefl, LIF receptor, Lin28, Nanog, Oct4, SoxlS, Sox2, Utfl, or a combination thereof 80. The in vitro culture of any one of embodiments 59-79, wherein the rat ES cells do not express one or more pluripotency s selected from c-Myc, Ecatl, Rexol, or a combination thereof 81. The in vitro culture of any one of ments 59-80, wherein the rat ES cells do not express one or more mesodermal markers ed from Brachyury, Bmpr2, or a combination thereof 82. The in vitro culture of any one of embodiments 59-81, wherein the rat ES cells do not express one or more endodermal markers selected from Gata6, Soxl7, Sox7, or combination thereof; 83. The in vitro culture of any one of embodiments 59-82, wherein the rat ES cells do not express one or more neural markers ed from Nestin, Pax6, or combination thereof 84. The in vitro culture of any one of embodiments 59-83, wherein the cell expresses a pluripotency marker comprising Oct-4, Sox2, alkaline phosphatase, or a combination thereof 85. The in vitro culture of any one of embodiments 59-84, wherein the rat ES cells are characterized by the expression of one or more of a rat ESC-specific gene selected from one or more of s Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-Al ), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gij), Insulin-like growth factor binding protein-like l (Igfbpll), Interleukin 36 beta(Illf8), Interleukin 28 receptor, alpha (Il28ra), left-right ination factor 1 (Leftyl), Leukemia inhibitory factor or alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), n tyrosine phosphatase non-receptor type 18 (Ptpnl 8), Caudal type homeobox 2 , Fibronectin type III and ankyrin repeat domains 1 (Fankl), Forkhead box El (thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box El (thyroid transcription factor 2) (Foxel), Hairy/enhancer-of— split related with YRPW motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lefl), Sal-like 3 (Drosophila) (Sall3), SATB homeobox l ), miR-632, or a combination thereof 86. The in vitro e of any one of embodiments 59-84, wherein the concentration of LIF is 50 U/ml to 150 U/ml. 87. The in vitro culture of any one of embodiments 59-85, wherein the concentration of LIF is 100 U/ml. 88. The in vitro culture of any one of embodiments 59-87, wherein the LIE is from mouse or comprises at least 92% sequence identity to SEQ ID NO: 1. 89. The in vitro culture of any one of embodiments 59-88, wherein the rat ES cell is capable of maintaining a pluripotency without requiring a paracrine LIF signaling. 90. The in vitro culture of any one of embodiments 59-89, wherein the feeder cell layer is not genetically modified to express LIF. 91. The in vitro culture of any one of embodiments 59-90, wherein the feeder cell layer comprises a monolayer of mitotically vated mouse embryonic fibroblasts (MEFs) 92. The in vitro culture of any one of embodiments 59-91, wherein the MEK inhibitor comprises PD032590l. 93. The in vitro culture of any one of embodiment 59-92, n the GSK-3 inhibitor comprises CHIR9902l. 94. The in vitro culture of any one of embodiments 59-93, wherein the population of rat ES cells is derived from a rat blastocyst-stage embryo or a rat morula-stage . 95. The in vitro culture of embodiment 94, wherein the blastocyst-stage or the morula-stage rat embryo r comprises an outgrowth of an amorphous undifferentiated mass of rat ES cells. 96. The in vitro culture of of embodiment 94, wherein the tion of rat ES cells comprises an isolated outgrth of an amorphous undifferentiated mass of rat ES cells. 2014/017452 97. A method for generating a rat embryonic stem (ES) cell line comprising: (a) culturing in vitro a first feeder cell layer and a morula or a blastocyst-stage rat embryo, wherein the zona pellucida of the morula or blastocyst-stage rat embryo has been removed, and n the culture ions maintain pluripotency of a rat ES cell and comprise a medium having mouse leukemia inhibitor factor (LIF) or a sequence having at least 91% sequence identity to SEQ ID NO:1 and having LIF activity, and a GSK3 inhibitor, and a MEK inhibitor; and, (b) erring an outgrowth of an amorphous undifferentiated mass of rat ES cells to an in vitro e well comprising a second feeder cell layer and culturing the outgrth under conditions comprising the medium having the mouse LIF or an active variant of the mouse LIF, and thereby maintaining pluripotency of the rat ES cells; and, establishing a rat ES cell line therefrom. 98. The method of embodiment 97, wherein the rat ES cell line is passaged at least 5 times. 99. The method of embodiment 97 or 98, wherein the rat ES cell line is passaged at least 10 times. 100. The method of embodiment 97, 98, or 99, wherein the medium comprises about 50 U/ml to about 150 U/ml of mouse LIF. 101. The method of any one of embodiments , wherein the medium comprises about 100 U/ml ofmouse LIF. 102. The method of any one of embodiments 97-101, wherein the feeder cell layer is not genetically modified to express LIF. 103. The method of any one of embodiments 97-102, wherein the feeder cell layer comprises a monolayer of mitotically inactivated mouse embryonic fibroblasts (MEFs). 104. The method of any one of embodiments 97-103, wherein the MEK tor comprises PD0325901. 105. The method of any one of embodiments 97-104, wherein the GSK-3 inhibitor comprises CHIR99021. 106. The method of any one of embodiment 97-105, wherein (a) the rat ES cell line is derived from an AC1 rat or derived from a Dark Agouti (DA) rat; (b) the rat ES cell line is derived from a -stage or a blastocyst-stage rat embryo; and/or, (c) the rat ES cell line is derived from a morula-stage or a blastocyst-stage embryo from a superovulated rat. 107. The method of any one of embodiments 97-106, wherein the medium r comprises at least one of an FGF receptor inhibitor, a ROCK inhibitor, or an ALK inhibitor. 108. The method of embodiment 107, wherein the FGF receptor inhibitor comprises PD184352, the ROCK inhibitor comprises Y-27632, or the ALK inhibitor comprises A01. 109. The method any one of embodiments 97-108, wherein at least one rat ES cell has a germline transmission efﬁciency of the targeted c modiﬁcation is at least 110. The method of embodiment any one of embodiments 97-109, wherein the germline transmission efﬁciency of the ed genetic modiﬁcation is at least 60%. 111. A method of selecting a rat embryonic stem (ES) cells having stably incorporated into its genome a heterologous polynucleotide comprising: (a) ing an in vitro tion of rat ES cells; (b) introducing into at least one rat ES cell a heterologous polynucleotide comprising a selection marker operably linked to a promoter active the rat ES cell; and, (c) culturing in vitro the rat ES cell population in an alternating ﬁrst and second e media, wherein the ﬁrst culture medium comprises an effective amount of a selection agent for a ﬁrst time period and the second culture medium does not comprise the selection agent, wherein the in vitro culture conditions are sufﬁcient to maintain pluripotency; thereby selecting the rat ES cell having stably integrated into its genome the heterologous polynucleotide. 112. The method of embodiment 111, n the ﬁrst and second culture media are alternated every 24 hours. 113. The method of embodiment 111 or 112, wherein the selection marker imparts ance to an antibiotic. 114. The method of any one of embodiments 111-113, wherein the antibiotic comprises G418. 115. The method of any one of embodiment 111-114, wherein the ion marker comprises neomycin phosphotransferase (neor), hygromycin B phosphotransferase (hygr), puromycin-N-acetyltransferase (puror) blasticidin S deaminase (bsrr), xanthine/guanine phosphoribosyl transferase (gpt), and herpes simplex virus thymidine kinase (HSV-k), or a ation thereof. 116. The method of any one of ments 111-115, wherein (a) the selection marker has an increased activity compared to the wild type ion marker; and/or (b) multiple copies of the selection marker are stably incorporated into the genome of the rat ES cell. 117. The method of embodiment 116, wherein the selection marker is a non- attenuated selection marker. 118. A method for genetically modifying an isolated rat embryonic stem (ES) cell comprising ucing into the genome of an isolated rat ES cell of any one of embodiment 1-58 a heterologous polynucleotide to form a genetically modiﬁed rat ES cell. 119. A method ofmaking a genetically modiﬁed rat comprising: (a) introducing into the genome of the isolated rat embryonic stem (ES) cell of any one of embodiments 1-58, a heterologous polynucleotide to form a rat ES cell having a genetic modiﬁcation; (b) introducing at least one of the rat ES cells comprising the targeted genetic modiﬁcation into a rat host embryo to produce an F0 embryo; (c) implanting the F0 embryo into a surrogate mother; (d) gestating the F0 embryo in the surrogate mother to term; and, (e) identifying an F0 rat haVing the targeted genetic modiﬁcation. 120. The method of embodiment 119, r comprising ng a male F0 rat with a wild type female rat to produce an Fl progeny that is heterozygous for the targeted c modiﬁcation. 121. The method of embodiment 120, further comprising breeding a male F0 rat with a wild type female rat to e an Fl y that is heterozygous for the targeted genetic modiﬁcation. 122. The method of embodiment 119, further comprising breeding a male rat of the F1 progeny with a female rat of the F1 progeny to obtain an F2 progeny that is homozygous for the c modiﬁcation. 123. The method of any one of embodiments 119-122, wherein at least 3% of the F0 rats haVing the c modiﬁcation transmit the genetic modiﬁcation to the F1 progeny. 124. The method of any one of embodiments 119-123, wherein at least 10% of the F0 rats haVing the c modiﬁcation transmit the genetic modiﬁcation to the F1 progeny. 125. The method of any one of ments 119-124, wherein at least 60% of the F0 rats having the genetic modiﬁcation transmit the genetic modiﬁcation to the F1 progeny. 126. The method of any one of embodiments 119-125, wherein the genetically modiﬁed rat ES cell is from the same rat strain as the rat host embryo. 127. The method of any one of embodiments 119-127, n the genetically modiﬁed rat ES cell is from a different rat strain as the rat host embryo. 128. The isolated population of rat ES cells of any of the preceding claims, the in Vitro culture of any of the preceding claims, or the method of any of the preceding claims, wherein the rat ES cells in the population se: (a) at least 90% of the rat ES cells are euploid; (b) at least 70% of the rat ES cells express at least one pluripotency marker; wherein the at least one pluripotency marker comprises Oct-4, Sox2, alkaline phosphatase, or a combination thereof; (c) a cell from the rat ES cell population, when combined with a rat host embryo transmits the genome of the rat ES cell line into an offspring; (d) the rat ES cells when cultured in Vitro loosely adhere to a feeder cell layer; (e) the rat ES cells form sphere-like colonies when plated on a feeder cell layer in Vitro; (f) the rat ES cells maintain pluripotency when cultured in Vitro in a media comprising an GSK3 inhibitor, a MEK inhibitor, LIF and a feeder cell layer that is not cally modiﬁed to express LIF; (g) the rat ES cell exhibits a targeting efﬁciency of homologous recombination of at least 2%; (h) the rat ES cells maintain pluripotency in Vitro without ing paracrine LIF signaling; (i) at least 70% of the rat ES cells are euploid and form sphere-like colonies when plated on a feeder cell layer in Vitro; (j) the rat ES cells s at least one pluripotency marker ed from Dnmt3L, Eras, ta, Fbxol5, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a ation thereof; (k) the rat ES cells do not express one or more differentiation markers selected from c-Myc, Ecat1, Rexol. (l) the rat ES cells do not express one or more mesodermal markers selected from Brachyury, Bmpr2, or a combination thereof; (m) the rat ES cells do not express one or more endomermal markers selected from Gata6, Soxl7, Sox7, or combination thereof; and/or (11) the rat ES cells do not express one or more neural markers ed from Nestin, Pax6, or combination thereof.

BRIEF DESCRIPTION OF THE FIGURES The patent or application file contains at least one drawing executed in color.

Copies of this patent or patent application publication with color g(s) will be ed by the Ofﬁce upon request and payment of the necessary fee. depicts rESCs, which grow as compact spherical colonies that routinely detach and ﬂoat in the dish. through D depicts various pluripotency markers expressed by rESCs: A depicts Oct-4 (green); B depicts Sox-2 (red); C s DAPI (blue); D depicts an y of pluripotency markers expressed by rESCs. depicts that the rESCs express light levels of alkaline phosphatase (a pluripotency ) (left), and the ype for line DA.2B is 42X,Y (right).

Karyotyping was done because rESCs often become tetraploid; lines were thus pre- screened by counting ase chromosome spreads, and lines with mostly normal counts were then formally karyotyped. depicts a closer view of a rESC of depicts production of chimeras by blastocyst injection and transmission of the rESC genome through the germline; chimeras produced by blastocyst ion using parental ACI.Gl rESC; high percentage chimeras usually have albino snouts. depicts Fl agouti pups with albino littermates, sired by ACI/SD chimera labeled with an asterisk (*) in Panel A depicts targeting of the rat Rosa 26 locus, which lies between the Setd5 and Thumpd3 genes as in mouse, with the same spacing. Panel A shows the structure of the mouse Rosa 26 locus. mRosa26 transcripts consist of 2 or 3 exons.

Panel B depicts the structure of the rRosa26 locus; the rat locus ns a second exon 1 (Exlb) in addition to the homologous exon to mouse exonl (Exla); no third exon has been identified in rat. Panel C depicts a targeted rRosa26 allele; homology arms of Skb each were cloned by PCR using genomic DNA from DA rESC; the ed allele contains a SA-lacZ-hUB-neo cassette ing a ll7bp deletion in the rRosa26 . depicts a control brain of a l4-week-old wild type rat, which was treated with X-gal. The control brain showed a low level of ound staining for LacZ (dorsal view). depicts LacZ expression in the brain of an rRosa26 heterozygous rat (14- week old). The lacZ reporter was expressed ubiquitously throughout the brain of the rRosa26 heterozygote. depicts a control heart and thymus (inset) of a l4-week-old wild type rat, which were treated with X-gal. The control heart and thymus showed a low level of background staining for LacZ. depicts LacZ expression in the heart and thymus (inset) of a 14-week- old rRosa26 heterozygous rat. The lacZ reporter was expressed ubiquitously throughout the heart and thymus of the rROSA26 heterozygote. depicts a l lung of a l4-week-old wild type rat, which were treated with X-gal. The control lung showed a low level of background staining for LacZ. depicts LacZ expression in the lung of a l4-week-old rRosa26 heterozygote rat. The lacZ reporter was expressed ubiquitously throughout the lung of the rRosa26 heterozygote. and H depict LacZ expression in e12.5 embryos. In contrast to the wild- type control embryo (H), which shows a low level of background LacZ staining, the rRosa26 heterozygous embryo exhibited ubiquitous expression of the LacZ reporter throughout the embryo. and J depict LacZ expression in el4.5 s. In st to the wild- type control embryo (J), which shows a low level of background LacZ staining, the rRosa26 zygous rat embryo exhibited ubiquitous expression of the LacZ reporter throughout the embryo.

Figure 9A-B provides a photograph showing the analysis of the chromosome number of the ACI.Gl rat ES cell line.

Figure lOA-B provides a photograph showing the analysis of the chromosome number of the DA.2B rat ES cell line.

Figure llA-B provides a photograph showing the is of the chromosome number of the DA.C2 rat ES cell line.

DETAILED DESCRIPTION The t methods and compositions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the methods and compositions are shown. Indeed, these methods and compositions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy able legal requirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the s and compositions set forth herein will come to mind to one skilled in the art to which this methods and compositions pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the methods and compositions are not to be limited to the specific embodiments disclosed and that modiﬁcations and other embodiments are included within the scope of the appended claims. Although specific terms are employed herein, they are used in a c and descriptive sense only and not for purposes of tion.

I. Overview The rat has long been the preferred rodent model organism for several fields of biomedical research such as cardiovascular e, metabolism, toxicology, neurobiology and behavior. Hundreds of strains of rat have been developed; some are excellent models for x human diseases such as hypertension, diabetes and cancer.

However, progress in understanding the genetics of these models has been severely hampered by the lty of modifying the rat genome in a controlled manner. Through the use of pecific endonucleases it is le to produce mutations in a gene of interest, but this method s imprecise and expensive. Targeting and germline transmission of rat ES cells remains a difficult task to achieve.

Isolation of rat ES cells (rESC) from two inbred s of rat is described herein. rESC from the DA and ACI strains were derived. These cells express pluripotency markers and exhibit a normal 42X,Y karyotype. High percentage chimeras have been produced, by microinjection into SD host embryos at the blastocyst stage, and transmission of the rESC genome has been demonstrated through the germline for both strains. Using plasmid targeting vectors, we have produced targeted ons in the rat lent of the ROSA26 locus, and we have achieved germline transmission of the targeted allele in both strains. These heterozygous animals express lacZ in all tissues at all stages examined.

In various aspects, ES cells were derived from the ACI strain in order to obtain a favorable number of male progeny from ACI donor ES cells. In one ment, the amount of male progeny is about 50%.

In various aspects, ES cells were derived from the DA strain in order to obtain primarily female progeny.

II. Rat Embryonic Stem (ES) Cells Various compositions and methods are provided herein which comprise nic stem (ES) cells from rat. Stem cells are a cell population possessing the capability to self-renew indeﬁnitely and are pluripotent. An "embryonic stem cell" or an “ES cell” comprises a stem cell obtained from an embryo or a fetus. The various rat ES cells provided herein can have one or more of any of the following properties: (a) have germ-line competency, meaning when the rat ES cell is implanted into a rat host embryo, the genome of the rat ES cell line is transmitted into an offspring; (b) have germ-line competency following at least one targeted genetic ation, meaning when the rat ES cell having the targeted genetic modiﬁcation is implanted into a rat host embryo, the targeted c modiﬁcation within the genome of the rat ES cell line is transmitted into an offspring; (c) have pluripotency in vitro; (d) have totipotency in vitro; (e) when cultured in vitro loosely adhere to a feeder cell layer; (f) when cultured in vitro form sphere-like es when plated on a feeder cell layer in vitro; (g) maintain pluripotency when cultured in vitro under conditions comprising a feeder cell layer that is not genetically modiﬁed to express leukemia inhibitor factor (LIF), wherein the culture media ses a ent concentration of LIF; (h) maintain pluripotency when cultured in vitro under conditions comprising a feeder cell layer, wherein the culture media comprises mouse LIF or an active t or nt thereof; WO 30706 (i) comprise a molecular signature that is characterized by i) the sion of one or more of rat ES cell-speciﬁc genes comprising Adheres Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-Al (Efnal), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gij), Insulin-like growth factor binding n-like l (Igfbpll), Interleulin 36 beta(Illf8), Interleukin 28 receptor, alpha (Il28ra), left-right determination factor 1 l), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2), Neuronal xin receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 (Ptpnl8), Caudal type homeobox 2 (Cdx2), Fibronectin type III and ankyrin repeat domains 1 (Fankl), Forkhead box El (thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box El id transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lefl ), Sal-like 3 phila) (Sall3), SATB homeobox l (Satbl), miR-632, or a combination thereof; ii) the expression ofat least 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, 14, 15, l6, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more of the rat ES cell-speciﬁc genes comprising Adheres Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent n kinase IV (Camk4), ephrin-Al (Efnal), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gij), n-like growth factor binding n-like l (Igfbpll), Interleulin 36 beta(Illf8), Interleukin 28 receptor, alpha (Il28ra), left-right determination factor 1 (Leftyl), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 (Ptpnl8), Caudal type homeobox 2 (Cdx2), Fibronectin type III and ankyrin repeat domains 1 (Fankl), Forkhead box El id transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box El (thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split d with YRPW motif 2 (Hey2), id enhancer-binding factor 1 (Lefl ), Sal-like 3 (Drosophila) (Sall3), SATB ox l (Satbl), 2, or a combination thereof; iii) at least a 20-fold increase in the expression of one or more of the rat ES cell-speciﬁc genes as set forth in Table 14 when compared to a F1H4 mouse ES cell; iv) at least a 20-fold increase in the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more ofthe rat ES cell-speciﬁc genes as set forth in Table 14 when compared to a F1H4 mouse ES cell; V) the expression of one or more of rat ES peciﬁc genes as set forth in Table 13; vi) the expression ofat least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more ofthe rat ES cell-speciﬁc genes as set forth in Table 13; vii) at least a 20-fold increase in the expression of one or more of the rat ES cell-speciﬁc genes as set forth in Table 13 when compared to a F1H4 mouse ES cell; viii) at least a 20-fold se in the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of the rat ES cell-specific genes as set forth in Table 13 when ed to a F1H4 mouse ES cell; ix) at least a 20-fold decrease in the expression of one or more of the rat ES cell-speciﬁc genes as set forth in Table 12 when ed to a F1H4 mouse ES cell; and/or x) at least a 20-fold decrease in the sion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of the rat ES cell-specific genes as set forth in Table 12 when compared to a F1H4 mouse ES cell; xi) any combination of sion of the rat ES cell-speciﬁc genes of parts (i)-(X); xii) a relative expression level of pluripotency markers as shown in Table 15 for at least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17 or 18 ofthe listed pluripotency markers. See, otency ranking column of Table 15 for relative expression levels; xiii) a relative expression level of the mesodermal markers as shown in Table 15 for at least 2, 3, or 4 of the listed mesodermal markers. See, mesodermal ranking column in Table 15 for relative expression levels; xiv) a relative expression level of endodermal markers as shown in Table for at least 2, 3, 4, 5, or 6 of the listed endodermal markers. See, endodermal ranking column in Table 15 for relative expression levels; xv) a relative sion level of neural markers as shown in Table 15 for at least 2 and 3 of the listed neural markers. See, neural ranking column in table 15 for relative expression levels; xvi) a relative expression level of ctoderm markers as shown in Table 15 for the listed trophectoderm markers. See, trophectoderm ranking column in table 15 for relative expression levels; xvii) any relative expression level of one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,2 4, 25, 26, 27, 28, 29 or 30) ofthe pluripotency markers, mesodermal markers, endodermal markers, neural markers and/or trophectoderm markers set forth in Table 15; xviii) the ve expression level of each of the markers set forth in table 15; xix) any combination of the signatures set forth in xii-xiix; and/or xx) any combination of the signature set forth in i-xiix; (j) have the ability to produce a F0 rat; (k) are capable of being subcultured and maintaining the undifferentiated state; (1) have the same number of somes as a normal rat cell; (m) maintain pluripotency in vitro without requiring paracrine LIF signaling; and/or (11) have self renewal, meaning they divide indefinitely while maintaining otency.

One or more of the characteristics outlined in (a)-(n) can be present in an isolated rat ES cell, a rat ES cell population or a rat ES cell line provided herein, wherein the rat ES cells have not undergone a ed genetic modification. In other embodiments, one or more of the characteristics outlined in (a)-(n) can be t in an ed rat ES cell, a rat ES cell population or a rat ES cell line provided herein that has one or more targeted genetic modifications. A targeted genetic modiﬁcation ses an alteration in the genome of the rat ES cell and includes, for example, an insertion, a deletion, a knockout, a knockin, a mutation, or a combination thereof. In other instances, the targeted genetic modiﬁcation ses at least one insertion of a logous polynucleotide into the genome of the rat ES cell. A ﬁarther description of such targeted genetic modiﬁcations are discussed elsewhere herein.

In c embodiments, the various rat ES cells and cell lines provided herein are germ-line ent, g when the rat ES cell is implanted into a rat host embryo, the genome of the rat ES cell is transmitted into an offspring. Such transmission into the offspring (i.e., the F1 population) can occur when the rat ES cell has not undergone a targeted genetic modiﬁcation. In addition, a rat ES cell haVing a targeted genetic modiﬁcation are also germ-line ent, meaning when the rat ES cell haVing the targeted genetic ation is implanted into a rat host embryo, the targeted genetic modiﬁcation of the rat ES cell is transmitted to the ing (i.e., the F1 population.) Thus, in various aspects, the rat ES cells and methods described herein are employed to obtain a high frequency, or high efﬁciency, of germline transmission of a a rat cell genome from both rat ES cells that have not undergone a targeted genetic modiﬁcation and also from rat ES cells that have undergone a targeted genetic modiﬁcation. In s embodiments, the ncy of germline transmission is greater than 1:600, greater than 1:500, greater than 1:400, greater than 1:300, greater than 1:200, and greater than 1:100. In various embodiments, the ncy of germline transmission is greater than 1%, greater than 2%, greater than 3%, r than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, up to about 16%, greater than 25%, greater than 50%, greater than 60%, greater than 65%, greater than 70%, greater than 75% or greater. In various embodiments, the frequency of germline transmission ranges from 9% to 16%. In various aspects, percent of donor rESC-derived progeny in the F1 generation is 1% or more, 2% or more, 3% or more, % or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, from 3% to about 10% or more; from 3% or more to about 63%, from about 10% to about 30%, from about 10% to about 50%, from about 30% to about 70%, from about 30% to about 60%, from about 20% to about 40%, from about 20% to 65%, or from about 40% to 70%. Thus, a rat ES cell provided herein that has not undergone a targeted genetic modiﬁcation or, alternatively, a rat ES cell that has a targeted genetic modiﬁcation have the ability to transmit their genome into the F1 population.

A rat ES cell that has not undergone a targeted genetic modiﬁcation or a rat ES cell that has a targeted genetic modiﬁcation can be pluripotent and/or totipotent.

“Pluripotent” or “pluripotency” refers to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm, mesoderm, or rm. Cell y is a l term which describes a cell's ability to differentiate into other cell types. See, for example, Hans et al. (2007). "The Potential of Stem Cells: An Inventory". Human biotechnology as Social Challenge. Ashgate Publishing, Ltd. p. 28, herein incorporate by reference. The term “totipotency” or “totipotent” is the ability of a single cell to divide and produce all of the differentiated cells in an organism. See, for example, Western P (2009). Int. J. Dev. Biol. 53 (2—3): 393—409, herein incorporated by reference. In speciﬁc embodiments, the various ES cells disclosed herein can be either pluripotent and/or totipotent. s methods can be used to determine if a rat ES cell is pluripotent. For e, the ES cell can be assayed for the expression of s pluripotent markers ing, but not limited to, Oct-4, Sox2, alkaline atase, or a combination thereof See, for example, Okamoto, K. et al., Cell, 60: 461-472 (1990), Scholer, H. R. et al., EMBO J. 9: 2185-2195 (1990)) and Nanog (Mitsui, K. et al., Cell, 113: 631-642 (2003), Chambers, I. et al., Cell, 113: 643-655 (2003) for various methods of assaying for the presence or the level of such markers. See, also Figures 2 and 3 provided herein.

Other otency markers include, for example, the presence of at least 1, 2, 3, 4, or 5 pluripotency marker comprising Nanog, Klf4, Dppa2, Fgf4, Rex1, Eras, ta and/or Sall3. Other pluripotency markers include, for example, the absence of at least 1, 2, 3, 4, , 6, 7, 8, 9 or 10 pluripotency marker comprising T/Brachyury, Flk1, Nodal, Bmp4, Bmp2, Gata6, Sox17, Hhex1, Sox7, and/or Pax6.

In speciﬁc embodiments, the expression and/or the level of expression of these markers can be determined using RT-PCR. Various kits are available to determine the level and/or presence of alkaline phosphatase, including, for example, an ALP tissue staining kit (Sigma) and Vector Red Alkaline Phosphatase Substrate Kit I (Funakoshi) and the like. Additional assays e in situ hybridization, immunohistochemistry, immunoﬁuorescence. In speciﬁc embodiments, the rat ES cell is characterized by expression of at least one pluripotency marker, including for example expression of Oct- 4, Sox2, alkaline phosphatase, or a combination f, and preferably all three of these markers.

The various rat ES cell provided herein (i.e. rat ES cells that have not undergone a targeted c modiﬁcation and/or rat ES cells that have a targeted genetic modiﬁcation) are capable of maintaining pluripotency and/or totipotency while being maintained in in vitro culturing conditions. Thus, the s rat ES cells provide herein can, in some embodiments, be subcultured while still maintaining the undifferentiated state. s methods of culturing the rat ES cells are discussed in further detail elsewhere herein.

The rat embryonic stem cells ed herein have been isolated from the rat embryo employing various isolation, puriﬁcation, and culture ion techniques which are sed in detail elsewhere . The term "cell" as used herein refers to individual cells, cell lines, or cultures derived from such cells. An “isolated” rat ES cell or rat embryo has been removed from its natural environment. The term ted” can mean free from 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the constituents with which a component is found in its l state. As used herein, a rat ES “cell line” comprises a population of isolated rat cells that were developed from a single rat ES cell and therefore the population efcells within a given cell line have a unifei‘m genetic makeup ether than fer mutations er haryetjypic changes eccurring during propagation or during targeted genetic modificatiens, For example, as indicated elsewhere, the disclesed rat ES cells are characterized by a, high level (if euploidy. Nevertheless, in seine cell lines the level efeuplcidy is less than 100% due to hai'yetypie changes in prepagatien of the line frem a single cell. Moreover, a given population of rat ES cells can se at least 10 exp 3, 10 exp4, 10x104, 10x105, 10x106, 10x107, 10x108, 10x109, or 10x1010 cells or greater. Some cell populations have sufﬁcient cells to permit selection of a desired d cell but not an excessively greater number so as to reduce the possibility of mutations or karyotypic changes ping in the cell line. For example, some cell populations have 10exp3 to 10exp6 cells.

As discussed elsewhere herein, various s are provided for the targeted c modiﬁcation of a rat ES cell line. When such methods are carried out, at least one cell within a rat ES cell line contains the targeted genetic modiﬁcation. Through various culturing and/or ion techniques rat ES cell lines having one or more desired targeted genetic modiﬁcations are produced.

In speciﬁc embodiments, a rat ES cell, a population of rat ES cell or a rat ES cell line (that have not undergone a targeted c modiﬁcation and/or have a targeted genetic modiﬁcation) are euploid, and thus have a chromosome number that is an exact multiple of the haploid number. In further embodiment, a rat ES cell, a population of rat ES cells or a rat ES cell line (that have not undergone a targeted genetic modiﬁcation and/or have a targeted genetic modiﬁcation) are diploid, and thus have two haploid sets of homologous chromosomes. When referring to a rat ES cell population or a population of cells from a given population of rat ES cells or a rat ES cell line (that have not undergone a targeted c modiﬁcation and/or have a targeted genetic modiﬁcation), at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% ofthe cells with the given population or euploid and/or diploid. In other ces, when referring to a rat ES cell population or a population of cells from a given rat ES cell line (that have not undergone a targeted genetic modiﬁcation and/or have a targeted genetic modiﬁcation), at least about 50% to 95%, about 60% to 90%, about 60% to 95%, about 60% to 85%, about 60% to 80%, about 70% to 80%, about 70% to 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 100%, about 80% to about 95%, about 80% to about 90%, about 90% to about 100%, about 90% to about 99%, about 90% to about 98%, about 90% to about 97%, about 90% to about 95% of the cells within the given tion are euploid and/or diploid.

In still r embodiments, a rat ES cell, a population of rat ES cells or a rat ES cell line (that have not undergone a targeted genetic modiﬁcation and/or have a targeted genetic modiﬁcation) have 42 chromosomes. When referring to a rat ES cell tion or a population of cells from a given rat ES cell line (that have not undergone a targeted genetic modiﬁcation and/or have a targeted genetic modiﬁcation) at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells with the given population have 42 chromosomes. In other instances, when referring to a rat ES cell population or a population of cells from a given rat ES cell line (that have not undergone a targeted genetic modiﬁcation and/or have a targeted genetic modiﬁcation) at least about 50% to 95%, about 60% to 90%, about 60% to 95%, about 60% to 85%, about 60% to 80%, about 70% to 80%, about 70% to 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 100%, about 80% to about 95%, about 80% to about 90%, about 90% to about 100%, about 90% to about 99%, about 90% to about 98%, about 90% to about 97%, about 90% to about 95% of the cells within the given population have 42 chromosomes.

In further embodiments, a rat ES cell, a population of rat ES cells or a rat ES cell line (that have not undergone a ed genetic modiﬁcation and/or have a targeted genetic modiﬁcation) provided herein form sphere-like colonies when plated on a feeder cell layer in vitro. The "sphere-like" morphology refers to the shape of rat ES cell colonies in culture, rather than the shape of individual ES cells. The rat ES cell colonies are spherical-like. Colonies which are loosely attached to the feeder cells appear ar (have a circular-like logy). Free-ﬂoating colonies are spherical-like. The rat ES cell colonies are spherical-like and very compact, g: the boundaries between cells are very hard to see. The edge of the colony appears bright and sharp. Individual nuclei are difﬁcult to distinguish because the cells are very small (so that the nucleus takes up most of the volume of the cell). Mouse ES Cells form elongated colonies and attach strongly to feeder cells. mESC morphology can vary with strain; e. g. B6 colonies are rounder and more domed than FlH4 colonies but are still more elongated than rESC.

Human ES cell colonies are ﬂatter and more spread out than mESC colonies. The instant rat ES colonies are not ﬂat and do not resemble human ES cell colonies.

In still further embodiments, a rat ES cell, a population of rat ES cells or a rat ES cell line (that have not undergone a targeted c modiﬁcation and/or have a targeted genetic modiﬁcation) have a circular morphology. A morphology scale for a circle is ed below, where a score of a 10 represents a perfect circle and a score of a 1 represents an ellipse.

Morphology scale of a circle: =A circle with a structure having a longitudinal axis and a vertical axis that run through the center of the structure and are of equal length. 9 = A structure having a longitudinal axis and vertical axis that run through the center of the structure, wherein one of the axis is between 0.9999 to 0.9357 the length of the other axis. 8 = A structure having a longitudinal axis and al axis that run h the center of the structure, wherein one of the axis is n 0.9357 to 0.875 the length of the other axis. 7 = A ure having a longitudinal axis and vertical axis that run through the center of the structure, wherein one of the axis is between 0.875 to about 0.8125 the length of the other axis. 6 = A structure having a longitudinal axis and vertical axis that run h the center of the structure, wherein one of the axis is between 0.8125 to 0.750 the length of the other axis.

= A structure having a longitudinal axis and vertical axis that run through the center of the ure, wherein one of the axis is between 0.750 to 0.6875 the length of the other axis. 4 =A structure having a longitudinal axis and vertical axis that run through the center of the structure, wherein one of the axis is between 0.6875 to 0.625 the length of the other axis.

WO 30706 3 =A structure having a longitudinal axis and vertical axis that run h the center of the structure, wherein one of the axis is between 0.625 to 0.5625 the length of the other axis. 2: A structure having a longitudinal axis and vertical axis that run h the center of the circle, wherein one of the axis is between 0.5625 to 0.523 the length of the other axis. 1: An e is deﬁned as having a longitudinal axis and al axis that run through the center of the structure, wherein one of the axis is between 0.523 to 0.500 the length of the other axis.

In one non-limiting embodiment, the rat ES cell population or a population of cells from a given rat ES cell line (that have not undergone a targeted genetic modiﬁcation and/or have a targeted genetic modiﬁcation) have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells with the given population have a circular morphology score of a 10, 9 or 8. In other embodiments, the rat ES cell population or a population of cells from a given rat ES cell line (that have not undergone a targeted genetic modiﬁcation and/or have a targeted genetic modiﬁcation) have at least about 50% to 95%, about 60% to 90%, about 60% to 95%, about 60% to 85%, about 60% to 80%, about 70% to 80%, about 70% to 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 100%, about 80% to about 95%, about 80% to about 90%, about 90% to about 100%, about 90% to about 99%, about 90% to about 98%, about 90% to about 97%, about 90% to about 95% of the cells within the given tion have a circular morphology score of a 10, 9, or 8.

In another non-limiting embodiment, the rat ES cell population or a population of cells from a given rat ES cell line (that have not undergone a targeted genetic modiﬁcation and/or have a targeted genetic modiﬁcation) have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells with the given population have a circular morphology score of a 7, 6, 5, 4 or 3. In other non-limiting embodiments, the rat ES cell population or a population of cells from a given rat ES cell line (that have not one a targeted genetic modiﬁcation and/or have a targeted genetic modiﬁcation) have at least about 50% to 95%, about 60% to 90%, about 60% to 95%, about 60% to 85%, about 60% to 80%, about 70% to 80%, about 70% to 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 100%, about 80% to about 95%, about 80% to about 90%, about 90% to about 100%, about 90% to about 99%, about 90% to about 98%, about 90% to about 97%, about 90% to about 95% of the cells within the given population have a circular morphology score of a 7, 6, 5, 4, or 3.

In still further embodiments, -like colonies form when the rat ES cells (that have not undergone a targeted genetic modiﬁcation and/or have a targeted genetic modiﬁcation) are plated on a feeder cell layer in vitro. A morphology scale for a sphere is ed below, where a score of a 10 represents a perfect sphere and a score of a 1 represents a three dimensional elliptical structure.

Morphology scale of a sphere-like ure: 10=A sphere is a structure haVing an X-axis and a Y-axis and a Z-axis each of which runs through the center of the structure and are of equal . 9 = A ure haVing an X axis and a Y-axis and a Z-axis that run through the center of the structure, wherein one of the axis is between 0.9999 to 0.9357 the length of at least one of the other axes. 8 = A structure haVing an X axis and a Y-axis and a Z-axis that run through the center of the structure, wherein one of the axis is between 0.9357 to 0.875 the length of at least one or both of the other axes. 7 = A ure haVing an X axis and a Y-axis and a Z-axis that run through the center of the structure, wherein one of the axis is between 0.875 to 0.8125 the length of at least one or both of the other axes. 6 =A structure haVing an X axis and a Y-axis and a Z-axis that run through the center of the structure, wherein one of the axis is between 0.8125 to 0.750 the length of at least one or both of the other axes.

= A structure haVing an X axis and a Y-axis and a Z-axis that run through the center of the structure, wherein one of the axis is 0.750 to 0.6875 the length of at least one or both of the other axes. 4 = A structure haVing an X axis and a Y-axis and a Z-axis that run through the center of the structure, wherein one of the axis is 0.6875 to 0.625 the length of at least one or both of the other axes. 3 =A structure haVing an X axis and a Y-axis and a Z-axis that run through the center of the structure, wherein one of the axis is between 0.625 to 0.5625 the length of at least one or both of the other axes. 2: A structure having an X axis and a Y-axis and a Z-axis that run through the center of the structure, wherein one of the axis is between 0.5625 to 0.523 the length of at least one or both of the other axes. 1: A structure having an X axis and a Y-axis and a Z-axis that run through the center of the ure, wherein one of the axis is between 0.523 to 0.500 the length of at least one or both of the other axes.

In one non-limiting embodiment, the rat ES cell population or a population of cells from a given rat ES cell line (that have not undergone a targeted genetic ation and/or have a targeted genetic ation) have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the colonies that form when the cells are plated on a feeder cell layer in vitro have a sphere-like morphology of a 10, 9 or 8. In other embodiments, the rat ES cell population or a population of cells from a given rat ES cell line (that have not one a targeted genetic modiﬁcation and/or have a targeted genetic modiﬁcation) have at least about 50% to 95%, about 60% to 90%, about 60% to 95%, about 60% to 85%, about 60% to 80%, about 70% to 80%, about 70% to 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 100%, about 80% to about 95%, about 80% to about 90%, about 90% to about 100%, about 90% to about 99%, about 90% to about 98%, about 90% to about 97%, about 90% to about 95% of the colonies that form when the cells are plated on a feeder cell layer in vitro have a sphere-like morphology of a 10, 9 or 8.

In another non-limiting embodiment, the rat ES cell population or a population of cells from a given rat ES cell line (that have not undergone a targeted genetic modiﬁcation and/or have a targeted genetic modiﬁcation) have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the colonies that form when the cells are plated on a feeder cell layer in vitro have a sphere-like logy of a 7, 6, 5, 4, or 3. In other embodiments, the rat ES cell population or a population of cells from a given rat ES cell line (that have not one a targeted genetic modiﬁcation and/or have a targeted genetic modiﬁcation) have at least about 50% to 95%, about 60% to 90%, about 60% to 95%, about 60% to 85%, about 60% to 80%, about 70% to 80%, about 70% to 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 100%, about 80% to about 95%, about 80% to about 90%, about 90% to about 100%, about 90% to about 99%, about 90% to about 98%, about 90% to about 97%, about 90% to about 95% of the colonies that form when the cells are plated on a feeder cell layer in vitro have a sphere-like logy of a 7, 6, 5, 4, or 3.

A given rat ES cell, a tion of rat ES cells or a rat ES cell line provided herein can be a male (XY) rat ES cell, a male (XY) population of rat ES cells, or a male (XY) rat ES cell line. In other embodiments, a population of rat ES cells or a rat ES cell line provided herein can be a female (XX) rat ES cell, a female (XX) population of rat ES cells, or a female (XX) rat ES cell line. Any such rat ES cell, population of rat ES cells or rat ES cell line can comprise the dy and/or diploidy as described above.

The various rat ES cell provided herein can be from any rat strain, including but not limited to, an ACI rat strain, a Dark Agouti (DA) rat strain, a Wistar rat strain, a LEA rat strain, a Sprague Dawley (SD) rat strain, or a Fischer rat strain such as Fisher F344 or Fisher F6. The various rat ES cells can also be obtained from a strain derived from a mix of two or more strains recited above. In one embodiment, the rat ES cell is derived from a strain selected from a DA strain and an ACI strain. In a specific embodiment, the rat ES cell is d from an ACI strain. The ACI rat strain is are characterized as having black agouti, with white belly and feet and an RT1 “V1 haplotype. Such strains are available from a variety of sources including Harlan Laboratories. In other embodiments, the various rat ES cells are from a Dark Agouti (DA) rat strain which is characterized as having an agouti coat and an RT1 “V1 haplotype. Such rats are ble from a variety of source including Charles River and Harlan Laboratories. In a r embodiment, the various rat ES cells provided herein are from an inbred rat strain.

In specific embodiments the rat ES cell line is from an ACI rat and comprises the ACI.Gl rat ES cell as described herein. In another embodiment, the rat ES cell line is from a DA rat and comprises the DA.2B rat ES cell line or the DA.2C rat ES cell line as bed herein.

A given rat ES cell provided herein can be obtained from a rat embryo at any stage of rat embryo development. Representative stages of rat embryo development are outline below in Table l. The rat embryos employed to derive the rat ES cells can be a - stage embryo, a blastocyst-stage , or a rat embryo at a developmental stage between a morula-stage embryo and a blastocyst-stage embryo. Thus, in speciﬁc embodiments, the rat embryo employed is at or between the Witschi stages of 5 and 7.

In other embodiments, the rat embryo employed is at the Witschi stage 5, 6, or 7.

In one embodiment, the rat ES cell is obtained from a rat blastocyst. In other embodiments, the rat ES cell is obtained from a cyst from a superovulated rat. In other embodiments, the rat ES cells are obtained from an 8-cell stage embryo which is then cultured in vitro until it develops into a morula-stage, blastocyst stage, an embryo between the Witschi stages 5 and 7, or into an embryo at the Witschi stage 5, 6, or 7. At which time the embryos are then plated. Morula-stage s comprise a t ball of cells with no internal cavity. Blastocyst-stage embryos have a visible internal cavity (the coel) and contain an inner cell mass (ICM). The ICM cells form ES cells.

Table 1. Stages of Rat Embryo pment Standard Stages Age Identiﬁcation of Stages. .

(Witschi) (days) Cleavage and Blastula l l 1 cell (in oviduct) 2 2 2 cells (in oviduct) 3 3 4 cells (in oviduct) 4 3.25 8-12 cells (in oviduct) 3.5 Morula (in uterus) 6 4 Early blastocyst (in uterus) 7 5 Free blastocyst (in uterus) Gastrula Implanting blastocyst, with trophoblastic cone and inner 8 6 cell mass; outgrowth of endoderm (hypoblast) rophoblast; inner cell mass nt), covered with 9 6.75 endoderm Near complete implantation; pendant begins differentiation 1 0 7 .25 . . . 1nto embryonic and extra-embryonic parts Completion of implantation; primary amniotic cyst; 11 7 75' ectoplacental cone Further provided are various rat ES cells (that have not undergone a ed genetic modiﬁcation and/or have a targeted genetic modiﬁcation) which are characterized by The rat ES cell is characterized by: i) the sion of one or more of rat ES cell-speciﬁc genes comprising Adheres Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-Al (Efnal), EPH receptor A4 ), gap junction protein beta 5 (Gjb5), Insulin-like growth factor binding protein-like l (Igfbpll), Interleulin 36 beta(Illf8), Interleukin 28 or, alpha (Il28ra), left-right determination factor 1 (Leftyl), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 (Ptpnl8), Caudal type homeobox 2 (Cdx2), ectin type III and ankyrin repeat domains 1 (Fankl), Forkhead box El (thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), ad box El (thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2 , Lymphoid enhancer-binding factor 1 (Lefl ), Sal-like 3 (Drosophila) (Sall3), SATB homeobox l (Satbl), miR-632, or a combination f; ii) the expression ofat least 2, 3, 4, 5, 6, 7, 8, 9,10,ll,l2,l3,l4,l5,l6,l7,l8, 19, 20, 21, 22, 23, 24, 25 or more of the rat ES cell-speciﬁc genes comprising Adheres ons Associate Protein (Ajapl), Claudin 5 (Cldn5), Cdc42 guanine tide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-Al (Efnal), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gjb5), n-like grth factor binding protein-like l (Igfbpll), Interleulin 36 beta(Illf8), Interleukin 28 or, alpha (Il28ra), left-right determination factor 1 (Leftyl), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2), al pentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 (Ptpnl8), Caudal type homeobox 2 (Cdx2), Fibronectin type III and ankyrin repeat domains 1 ), ad box El (thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box El (thyroid transcription factor 2) (Foxel), enhancer-of-split d with YRPW motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lefl), Sal-like 3 (Drosophila) (Sall3), SATB homeobox l (Satbl), miR-632, or a combination thereof; iii) at least a 20-fold increase in the expression of one or more of the rat ES cell- speciﬁc genes as set forth in Table 14 when compared to a FlH4 mouse ES cell; iV) at least a 20-fold increase in the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, l4, l5, l6, l7, l8, 19, 20, 21, 22, 23, 24, 25 or more ofthe rat ES cell-specific genes as set forth in Table 14 when compared to a FlH4 mouse ES cell; V) the expression of one or more of rat ES cell-specific genes as set forth in Table 13; Vi) the sion ofat least 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, l4, l5, l6, l7, l8, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more ofthe rat ES cell-speciﬁc genes as set forth in Table 13; vii) at least a 20-fold increase in the sion of one or more of the rat ES cell- speciﬁc genes as set forth in Table 13 when compared to a F1H4 mouse ES cell; viii) at least a 20-fold increase in the sion of at least 2, 3, 4, 5, 6, 7, 8, 9, , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of the rat ES cell-speciﬁc genes as set forth in Table 13 when compared to a F1H4 mouse ES cell; ix) at least a 20-fold decrease in the sion of one or more of the rat ES cell- specific genes as set forth in Table 12 when compared to a F1H4 mouse ES cell; and/or x) at least a 20-fold decrease in the sion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more ofthe rat ES cell-specific genes as set forth in Table 12 when compared to a F1H4 mouse ES cell; xi) any ation of expression of the rat ES cell-specific genes of parts (i)- (X); xii) a relative expression level of pluripotency markers as shown in Table 15 for at least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17 or 18 ofthe listed otency markers. See, otency ranking column of Table 15 for relative expression levels; xiii) a relative expression level of the mesodermal markers as shown in Table 15 for at least 2, 3, or 4 of the listed mesodermal markers. See, mesodermal ranking column in Table 15 for relative expression levels; xiv) a relative expression level of endodermal markers as shown in Table 15 for at least 2, 3, 4, 5, or 6 of the listed endodermal markers. See, endodermal ranking column in Table 15 for relative expression levels; xv) a relative expression level of neural s as shown in Table 15 for at least 2 and 3 of the listed neural markers. See, neural ranking column in table 15 for relative expression levels; xvi) a relative expression level of trophectoderm markers as shown in Table 15 for the listed trophectoderm markers. See, trophectoderm ranking column in table 15 for relative expression levels; xvii) any relative expression level of one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,2 4, 25, 26, 27, 28, 29 or 30) ofthe pluripotency s, mesodermal markers, endodermal markers, neural markers and/or trophectoderm markers set forth in Table 15; xviii) the relative expression level of each of the markers set forth in table 15; xix) any combination of the signatures set forth in xii-xiix; and/or xx) any combination of the signature set forth in i-xiix.

In one embodiment, when transplanted into a pre-morula stage rat embryo, the rat ES cell (that have not undergone a ed genetic modiﬁcation and/or have a targeted genetic modiﬁcation) can contribute to at least 90% of the cells in an F0 generation, contribute to at least 95% of the cells in an F0 generation, bute to at least 96% of the cells in an F0 generation, contribute to at least 97% of the cells in an F0 generation, contribute to at least 98% of the cells in an F0 generation, or bute to at least 99% of the cells in an F0 generation.

III. Derivation and Propagation ofRat Embryonic Stem (ES) Cells Various s are provided for obtaining the rat ES cells disclosed . In speciﬁc embodiments, such methods comprise (a) providing an in vitro culture comprising a feeder cell layer and a population of isolated rat embryonic stem (ES) cells; (b) culturing in vitro under conditions which are sufﬁcient to maintain pluipotency and/or totipotency of the isolated rat ES cell. Such methods thereby allow for the propagation of a rat ES cell population and/or a rat ES cell line.

In one embodiment, a method for culturing a rat embryonic stem cell line is provided. Such methods comprises culturing in vitro a feeder cell layer and a rat ES cell line, wherein the culture conditions maintain pluripotency of the rat ES cells and comprise a media having mouse leukemia inhibitor factor (LIF) or an active variant or fragment thereof. The various methods ﬁarther se passaging and culturing in vitro the cells of the rat ES cell line, wherein each subsequent in vitro culturing ses ing the rat ES cells on the feeder cell layer under conditions that maintain pluripotency of the rat ES cells and comprises a media having mouse LIF or an active variant or fragment thereof. i. Culture Conditions The culture media ed in the various methods and compositions will maintain the rat ES cells. The terms "maintaining" and "maintenance" refer to the stable preservation of at least one or more of the characteristics or ypes of the rat ES cells outline herein. Such phenotypes can include maintaining pluripotency and/or totipotency, cell morphology, gene expression s and the other ﬁlnctional teristics of the rat stem cells described herein. The term "maintain" can also encompass the propagation of stem cells, or an increase in the number of stem cells being cultured. The term ﬁlrther contemplates culture conditions that permit the stem cells to remain pluripotent, while the stem cells may or may not continue to divide and increase in number.

The term "feeder cell" or “feeder cell layer” refers to a e of cells that grow in vitro and secrete at least one factor into the culture medium that is used to support the growth of another cell of interest in the culture. The feeder cells employed herein aid in maintaining the otency of the rat ES cells, and in speciﬁc embodiments, one or more of the other characteristics or phenotypes described . Various feeder cells can be used including, for example, mouse embryonic asts, including mouse embryonic ﬁbroblasts obtained between the 12th and 16th day of pregnancy. In speciﬁc embodiments, feeder cell layer comprises a monolayer of mitotically inactivated mouse embryonic ﬁbroblasts (MEFs).

The in vitro cultures of the rat ES cells ﬁarther comprise an effective amount of Leukemia Inhibitor Factor (LIF) or an active variant or fragment thereof. Leukemia inhibitory factor (LIF) belongs to the IL-6 receptor family. LIF binds to a heterodimeric membrane receptor made up of a LIF-speciﬁc subunit, gp190 or LIFR, and the subunit gp130, which is shared with the other members of the IL-6 family. LIF inhibits the differentiation of embryonic stem cells in mice and contribute to stem cell self-renewal.

Human and mouse LIF share 79% sequence homology and exhibit cross-species activity.

Rat LIF (rtLIF) is a 22.1 kDa protein containing 202 amino acid es that exhibits 91% amino acid sequence identity with murine LIF ama et al. 1998). There are six possible asparagine-linked glycosylation (N-glycosylation) sites which are conserved among the LIF ptide from the various species and an additional site of Asn150 which is speciﬁc for rat LIF. The tertiary structure of the mouse LIF and its ﬁlnction is bed in further detail in Aikawa et al. (1998) Biosci. Biotechnol. m. 62 1318- 1325 and k et al. (2005) Immunology nancy, editor Gil Mor., US Patent No. 5,750,654 and D P g (1987) EMBO Journal 198720, each ofwhich is herein incorporated by reference in their entirety. A partial mouse LIF sequence is reported on the SwissProt website under the accession number P09056.

Mouse LIF activity is assessed by its ability to induce differentiation of M1 myeloid leukemia cells. The speciﬁc activity is 1 x 106 units/ml (Cat. No. 03-0011 from Stemgent) and 1 x 107 units/ml (Cat. No. 03100 from Stemgent), where 50 units is deﬁned as the amount of mouse LIF required to induce differentiation in 50% of the M1 colonies in 1 ml of medium. See, also, Williams, R.L. et al. (1988) Nature 336: 684- 687.; Metcalf, D. et al. (1988) Leukemia 2: 1; Niwa, H. et al. (2009) Nature 460: 118-122; Xu, J. et al. (2010) Cell Biol Int. 34: 791-797; Fukunaga, N. et al. (2010) Cell Reprogram. 12: 369-376; and, Metcalf D. (2003) Stem Cells 21: 5-14, each of which is herein incorporated by reference in their entirety. An “effective amount of LIF” comprises a concentration of LIF that allows the rat ES cells of an in vitro e to remain in an erentiated gv'lui'lpetent state. Vatli‘ms markers that can be used, t0 assay for the cells remaining in a plurlpetent state are sed ere herein, The Lil: petypeptlde en'ipleyed in the writing i'netheds and een‘zpi‘mitiens previded herein can be from any organism, including from a mammal, a rodent, a human, a rat or a mouse. In one embodiment, the LIE polypeptide is from a mouse. In still r embodiments, the mouse LIF polypeptide comprises the amino acid sequence set forth in SwissProt Accesion number: P09056, which is herein incorporated by reference in its entirety and is also set forth in SEQ ID NO: 1.

In other embodiments, an active t or fragment of the mouse LIF ptide as set forth in SEQ ID NO: 1 or in SwissProt Accesion number: P09056 can be used. Such active variants and fragments (including active variants having at least 75%, 80%, 85% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1) are discussed in further detail elsewhere .

LIF polypeptide or the active t or fragment thereof can be provided to the in vitro culture in a variety of ways. In one embodiment, the effective amount of the LIE polypeptide or the active variant or fragment thereof is added to the culture media. In other ments, the feeder cells have been genetically modiﬁed to press the LIE polypeptide or the active variant or fragment thereof. Such feeder cells include feeder cells prepared from gamma—irradiated or mlten‘ayelwl? treated BIA—M meme fibroblasts that express metrixnasseeieted LIE. Method of generating and using such genetically modiﬁed feeder cells can be found, for example, in See, Buelir er al. 2003) Bit)! Repma’ 682222429, Rathjen er a}. (T1990) Cell 62 llllﬁwl l 15, and Buehr er al. (2008) Cell 1315:12874298“, each el‘whlcb is herein ineerpm‘eted by reference The heterologous LIF expressed in the feeder cells can be from the same organism as the feeder cells or from an organism that is different from that of the feeder cell. In addition, the heterologous LIF expressed in the feeder cells can be from the same or from a different organism than the ES cells the feeder layer is supporting.

In still other embodiments, the feeder cells employed in the various methods disclosed herein are not genetically modiﬁed to express a heterologous LIF polypeptide or an active variant or fragment thereof. Thus, in particular embodiments, the monolayer of mitotically inactivated mouse embryonic ﬁbroblast employed in the methods has not been cally modiﬁed to express a heterologous LIF ptide.

In other embodiments, the LIE polypeptide or the active variant or fragment thereof is added to the culture media. When LIF is added to the culture media, the LIE can be from any organism, ing from a mammal, a rodent, a human, a rat or a mouse. In one embodiment, the LIE present in the e media is from a mouse. In still ﬁarther embodiments, the mouse LIF polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In other embodiments, an active variant or fragment of the mouse LIF polypeptide as set forth in SEQ ID NO:l can be used. Such active variants and fragments ding active ts having at least 75%, 80%, 85% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1) are discussed in further detail elsewhere herein.

In speciﬁc embodiments, the rat ES cells and rat ES cell lines provided herein maintain pluripotency in vitro Without requiring paracrine LIF signaling.

In speciﬁc embodiments, LIF or an active variant or fragment thereof is present in the culture media at any concentration that maintains the rat ES cells. LIF polypeptide or active variant or fragment thereof is present in the culture media at about 25U/ml to about 50U/ml, at about 50U/ml to about lOOU/ml, at about lOOU/ml to about 125U/ml, at about 125U/ml to about l, at about l50U/ml to about l75U/ml, at about l to about 200U/ml, at about 200U/ml to about 225U/ml, at about 225U/ml to about 250U/ml, at about 250U/ml to about 300U/ml, to about 300U/ml to about 325U/ml, at about l to about 350U/ml, at about 350U/ml to about 400U/ml, at about 400U/ml to about 425U/ml, at about 425U/ml to about 450U/ml, at about 450U/ml to about 475U/ml, at about 475U/ml to about 500U/ml, at about 75U/ml to about 500U/ml or greater. In other embodiments, LIF polypeptide or active variant or fragment thereof is present in the e media at about 25U/ml to about 50U/ml, at about 25U/ml to about lOOU/ml, at about 75U/ml to about 125U/ml, at about 50U/ml to about l50U/ml, at about 90U/ml to about 125U/ml, at about 90U/ml to about llOU/ml, at about 80U/ml to about l50U/ml, at about 80U/ml to about 125U/ml. In a c embodiment, LIF ptide or active variant or fragment f is present in the culture media at about lOOU/ml.

When mouse LIF is employed, the mouse LIF polypeptide or active variant or fragment thereof is present in the culture media at any concentration that maintains the rat ES cells. Mouse LIF polypeptide or active variant or fragment f is present at about 25U/ml to about 50U/ml, at about 50U/ml to about lOOU/ml, at about lOOU/ml to about 125U/ml, at about 125U/ml to about l50U/ml, at about l50U/ml to about l75U/ml, at about l75U/ml to about 200U/ml, at about 200U/ml to about 225U/ml, at about 225U/ml to about 250U/ml, at about 250U/ml to about l, to about 300U/ml to about 325U/ml, at about l to about 350U/ml, at about l to about 400U/ml, at about 400U/ml to about 425U/ml, at about 425U/ml to about 450U/ml, at about 450U/ml to about 475U/ml, at about l to about 500U/ml, at about 75U/ml to about l or greater. In other embodiments, mouse LIF polypeptide or active variant or fragment thereof is present at about 25U/ml to about 50U/ml, at about 25U/ml to about lOOU/ml, at about 75U/ml to about 125U/ml, at about 50U/ml to about l50U/ml, at about 90U/ml to about 125U/ml, at about 90U/ml to about llOU/ml, at about 80U/ml to about l50U/ml, at about 80U/ml to about l. In a specific embodiment, mouse LIF polypeptide or active variant or fragment thereof is present in the culture media at about l.

The culture media employed maintains rat ES cells. As such, in specific embodiments, the culture media employed in the various method and compositions will maintain the pluripotency of all or most of (i.e., over 50%) of the rat ES cells in a cell line for a period of a at least 5, 10 or 15 passages. In one embodiment, the culture media comprises one or more compounds that assist in maintaining pluripotency. In one embodiment, the culture media comprises a MEK pathway inhibitor and a glycogen synthase kinase-3 ) inhibitor. The media can further comprise additional ents that aid in maintaining the ES cells, including for e, FGF or inhibitors, ROCK inhibitors, and/or ALK (TGFb receptor) inhibitors. A non-limiting example of an FGF receptor inhibitors includes PD184352. A non-limiting example of a ROCK inhibitor includes Y-27632, and non-limiting example of an ALK (TGFb receptor) inhibitor includes AOl. In speciﬁc embodiments, 2i media (table 2) is used with 10 uM ROCKi when thawing cryopreserved rESC or when re-plating rESC after dissociation with trypsin.

In other embodiments, the media ses a combination of inhibitors consisting of a MEK pathway inhibitor and a glycogen synthase kinase-3 ) inhibitor.

In one non-limiting embodiment, the culture media comprises a GSK-3 inhibitor comprising CHIR99021 and/or comprises a MEK inhibitor comprising 901. In other embodiments, the media comprises a combination of inhibitors consisting of CHIR99021 and PD0325901. Either of these compounds can be obtained, for example, from Stemgent. In speciﬁc embodiments, CHIR99021 is present in the culture media at a tration of about 0.5 u to about 3 uM, about 0.5u to about 3.5 uM, about 0.5 uM to about 4 uM, about 0.5uM to about 1 uM, about 1 uM to about l.5uM, about l.5uM to about 2 uM, about 2 uM to about 2.5 uM about 2.5 to about 3 uM, 3 uM to about 3.5 uM. In further embodiments, CHIR99021 is present in the culture media at a tration of about 3 uM. In other embodiments, PD0325901 is present in the culture media at a concentration of about 0.4 uM to about luM, about 0.4 uM to about 1.5 uM, about 0.4 uM to about 2 uM, about 0.4 uM to about 0.8 uM, 0.8uM to about 1.2 uM, about 1.2 to about 1.5 uM. In further ments, PD0325901 is present in the culture media at a concentration of about luM. In speciﬁc embodiments, CHIR99021 is present in the culture media at a concentration of about 3 uM and PD0325901 is present at a concentration of about luM.

In one non-limiting embodiment, the culture media employed in the various methods and itions sed herein is set forth in Table 2. Within the context of this application, the media described in Table 2 is referred to as 2i media.

Table 2: Non-limiting rat ES culture media.

Reagent DMEM/F l2 basal media asal media Penicillin/streptomycin L-Glutamine 2-Mercaptoethanol N2 supplement B27 supplement LIF 100 U/ml PD0325901 (MEK inhibitor).

CHIR99021 (GSK inhibitor).

Additional media that can be employed e those disclosed in Li et al. (2008) Cell 135:1299-1310, Yamamoto et al. (2012) Transgenic Rats 21:743-755, Ueda et al. (2008) PLoS ONE 2800, Meek et al. (2010) PLoS ONE 4 (l2): el4225; Tong et al. (2010) Nature 467:211-213; US Patent Publication 2012/0142092, Buehr et al. (2008) Cell l35:l287-l298, Li et al. (135) Cell 1299-1310, each ofwhich is herein incorporated by reference in their entirety. When employing such media, the concentration and the source of LIF can be d as outlined herein. In c ments, the various culture medias are used in combination with mouse LIF or an active variant or fragment thereof, and in even further embodiments, the s culture medias comprise a mouse LIF or an active variant or fragment thereof at a concentration of about 50U/ml to about 100U/ml, about 50U/ml to about l, or about 100U/ml.

The temperature of the cultures of rat ES cells, both for the production of the ES cell line and for the culturing and maintaining of the ES line it typically carried out at about 35°C to about 37.50 C. In speciﬁc embodiment, the temperature is 370°C. The culture is typically carried out at 7.5% C02. ii. Establishing a rat ES Cell Line Methods for generating a rat embryonic stem (ES) cell line are provided. Such methods comprises (a) culturing in vitro a ﬁrst feeder cell layer and a morula-stage embryo, a blastocyst-stage , or a rat embryo at a developmental stage between a -stage embryo and a blastocyst-stage embryo, wherein the zona pellucida of the rat embryo has been removed and wherein the culture ions maintain pluripotency of the rat ES cell and comprises a media having mouse leukemia inhibitor factor (LIF) or an active variant or fragment thereof; and, (b) transferring an outgrowth of an amorphous undifferentiated mass of rat ES cells to an in vitro culture well comprising a second feeder cell layer and culturing the outgrowth under conditions that maintain pluripotency of the rat ES cells and comprises a media having mouse LIF or an active variant or fragment thereof, and thereby establishing a rat ES cell line. The various methods r comprise passaging and ing in vitro the cells of the rat ES cell line, wherein each uent in vitro culturing comprises culturing the rat ES cells on the feeder cell layer under conditions that maintain pluripotency of the rat ES cells and comprises a media having mouse LIF or an active variant or fragment thereof Rat ES cell lines made by such methods are also provided.

Non-limiting examples of methods to establish a rat ES cell line having the s characteristics discussed herein are set forth in Example 3. Brieﬂy, a rat embryo (i.e., a morula-stage embryo, a blastocyst-stage embryo, or a rat embryo at a developmental stage between a morula-stage embryo and a blastocyst-stage embryo) is 2014/017452 ﬂushed from the uteruses of a female rat. In speciﬁc embodiments, a blastocyst or an 8 cell embryo is obtained. The zona pellucida is removed and the rat embryos are cultured on feeder cells (as sed elsewhere herein) which, in speciﬁc embodiments, comprise a monolayer of cally inactivated mouse embryonic ﬁbroblasts (MEFs). The cells of the morula-stage embryo, the cyst-stage embryo, or the rat embryo at a developmental stage between a morula-stage embryo and a blastocyst-stage embryo are cultured in vitro under conditions that maintain the ES rat cells and thereby are sufﬁcient to maintain pluripotency and/or totipotency of the ES cells. Various medias can be employed at this stage, ing any of the various medias discussed above which have LIF, including mouse LIF or an active t or fragment thereof, in the media.

The cultures are monitored for the presence of an outgrowth (an amorphous undifferentiated mass of . Once the outgrth reaches an appropriate size, a given outgrowth is transferred to new feeder plate and cultured. Transfer is accompanied by enzymatic dissociation using trypsin in order to produce le colonies. This transfer is commonly referred to as “passage 1”. The speed at which each line expands varies.

The media is changed as needed in order to maintain the pluripotency or totipotency of the rat ES cells. The culture is monitored for the presence of colonies having embryonic stem cell morphology. Such morphology includes one or more of the following characteristics: (a) round, circular mounds that rise above the monolayer of feeder cells; (b) cells that are packed tightly together such that cell boarders are difﬁcult to see; (c) smaller cell size; (d) small amount of cytoplasm and enlarged nucleus, (e) form sphere- like colonies when plated on feeder cells in vitro. Once such colonies appear, the culturing can continue until reaching approximately 50% ncy. The colonies are then transferred to a new feeder plate. er is accompanied by enzymatic dissociation using trypsin in order to expand the number of colonies. This is referred to as ge 2”. The cells are continued to be cultured with feeder cells until they reach approximately 50% conﬂuent, at which point the cells can undergo r passages to maintain the cell lines or the lines can be frozen. See, also Tong et al. (2010) Nature 467 (9):2l l-2l5; Li et al. (2008) Cell 135 : 1299-13 10, and Buehr et al. (2008) Cell 135: 1287-1298, each of which is herein incorporated by reference. Thus, in speciﬁc embodiments, the various rat ES cells, cell lines and cell populations disclosed herein are capable of being subcultured and maintaining the undifferentiated state.

In one miting embodiment, the derivation of the rat ES cells occurs as follows. At day 0, female rats are euthanized and the ts and uterine horns are dissected out and place into a tissue culture dish containing warm N2B27 media. Media is ﬂushed through the uterine horns and oviducts to eject blastocysts into the media. The blastocysts are collected and transfer to embryo culture dish containing KSOM + 2i (1 uMPD0325901, 3 uM CHIR9902l). KSOM can be sed from Millipore, catalog number is MRD. The 2i media referred to herein comprises the media set forth in Table 2. The cells are culture overnight at 37° at 7.5% C02.

In other non-limiting embodiments, the rat ES cells are derived from 8-cell embryos or frozen 8-cell embryos. The s are cultured in M2 medium for 10 s at room temperature and are then transfer to KSOM + 2i and culture overnight.

A non-limiting example for derivation of the rat ES cells is as follows: on Day 1, transferring cavitated embryos to 2i medium (Table 2) and culture overnight. The culturing is continued un-cavitated in KSOM +2i. On day 2, all remaining embryos are transferred to 2i medium, whether or not they have cavitated. Culture continues ght in 2i medium. On day 3, s are incubated with acid tyrodes to remove the zona pellucida and washed 3 times in 2i medium to remove the acid tyrodes. Each embryo is deposited into a separate well feeder plate, in which each well ns a monolayer of mitotically inactivated mouse embryonic fibroblasts (MEFs). The cells are cultured ght in 2i medium. On day 4 and 5, the cells plated embryos are monitored for the presence of an outgrowth, an amorphous undifferentiated mass of cells. Outgrowths are ready for transfer when they are approximately twice the size of the plated embryo. Each day, spent media is removed and replaced with fresh 2i media.

The outgrowths are transferred to new feeder wells, and again spent media is removed and the wells are washed with PBS. The PBS is removed and trypsin is added and incubated for about 10 minutes. The trypsin reaction is stopped by the addition of 30ul 2i media and 10% PBS. The cells are gently dissociated and the entire content is erred to a well in a feeder plate. This is referred to as Passage 1 (Pl). The cells are cultured overnight in 2i medium. On day 5-8, depending on how fast each line expands, the 2i media is changed each day and e is red for the ce of colonies with an ESC logy. Such ESC morphology is discussed in detail ere herein. Culturing continues until colonies expand to about 50% conﬂuency. The colonies are then trypsinzied and passaged as before into feeder wells. This is referred to as passage 2. Feeding and monitoring each line is continued until they are approximately 50% conﬂuent. The cells are trypsinized as usual. The trypsin is stopped with 2i media + 10% PBS. The cells are ed by centriﬁlgation, and the cells in 400 ul Freezing Medium (70% 2i, 20% PBS, 10% DMSO). The cells can then be frozen. This is referred to passage 3. iii. Maintaining and Passaging a rat ES cell Line Further provided are methods for maintaining or culturing a rat embryonic stem cell line. The method comprises culturing in vitro a feeder cell layer and a rat ES cell line, n the e conditions maintain pluripotency of a rat embryonic stem (ES) cell and comprise a media having mouse leukemia inhibitor factor (LIF) or an active variant or fragment thereof. Such methods employ the culture media and feeder cell layer as outlined above. In one embodiment, the rat ES cell line can be passaged at least , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50 or more times. In speciﬁc ments, the rat ES cells can be passaged up to at least 11 times in a medium comprising a GSK3 inhibitor and a MEK inhibitor without decreasing its targeting efﬁciency or germline transmission efﬁciency of a targeted genetic modiﬁcation.

The rat ES cell lines are fed and monitored. In speciﬁc embodiments, passage occurs when the culture is approximately 30%, 40%, 50%, or 60% conﬂuent. In other embodiments, passage occurs when the culture is 50% conﬂuent. ing on how fast each line expands, the es can occur every 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95 or 100 hours. In other embodiments, the time between passages ranges between 24 hours and 96 hours, between about 30 and 50 hours, between about 25 and 75 hours, between about 50 and 96 hours, between about 25 and 75 hours, between about 35 and 85 hours, or between about 40 and 70 hours. In one embodiment, the rat ES cell, cell line or cell population as disclosed herein has a doubling time ranging from about 24 hours to about 36 hours. In one embodiment, the rat ES cell has a doubling time of 25 hours.

The s rat ES cell lines when derived and maintained as outlined herein can have one or more any the following properties: (a) have germ-line competency, meaning when the rat ES cell is implanted into a rat host embryo, the genome of the rat ES cell line is transmitted into an offspring; (b) have germ-line competency following ed genetic modiﬁcation, meaning when the rat ES cell is implanted into a rat host embryo, the targeted genetic modiﬁcation within the genome of the rat ES cell line, is transmitted into an offspring; (c) have pluripotency in vitro; (d) have totipotency in vitro; (e) when cultured in vitro loosely adhere to a feeder cell layer; (f) when cultured in vitro form sphere-like colonies when plated on a feeder cell layer in vitro; (g) in pluripotency when cultured in vitro under conditions comprising a feeder cell layer that is not genetically modified to express leukemia inhibitor factor (LIF), wherein the e media comprises a sufficient concentration of LIF; (h) maintain pluripotency when cultured in vitro under conditions comprising a feeder cell layer, wherein the culture media comprises mouse LIF or an active variant or fragment thereof; (i) comprise a lar signature characterized by i) the expression of one or more of rat ES cell-speciﬁc genes comprising Adheres Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine nucleotide ge factor 9 (Arhgef9), m/calmodulin-dependent protein kinase IV (Camk4), ephrin-Al (Efnal), EPH or A4 (Epha4), gap junction protein beta 5 (Gij), Insulin-like growth factor binding n-like l ll), Interleulin 36 beta(Illf8), Interleukin 28 receptor, alpha (Il28ra), left-right determination factor 1 (Leftyl), Leukemia tory factor receptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III and ankyrin repeat s 1 (Fankl), Forkhead box El (thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), ad box El (thyroid transcription factor 2) ), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lefl ), Sal-like 3 (Drosophila) (Sall3), SATB homeobox l (Satbl), miR-632, or a combination thereof; ii) the expression ofat least 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, 14, 15, l6, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more of the rat ES cell-speciﬁc genes sing Adheres Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-Al ), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gjb5), Insulin-like growth factor binding protein-like 1 (Igfbpll), Interleulin 36 beta(Illf8), Interleukin 28 receptor, alpha (Il28ra), left-right determination factor 1 l), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III and ankyrin repeat domains 1 (Fankl), ad box E1 (thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2 , Lymphoid enhancer-binding factor 1 (Lefl ), Sal-like 3 (Drosophila) (Sall3), SATB homeobox 1 (Satbl), miR-632, or a combination thereof; iii) at least a 20-fold increase in the expression of one or more of the rat ES peciﬁc genes as set forth in Table 14 when compared to a FlH4 mouse ES cell; iV) at least a 20-fold increase in the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more ofthe rat ES cell-speciﬁc genes as set forth in Table 14 when compared to a FlH4 mouse ES cell; V) the expression of one or more of rat ES cell-speciﬁc genes as set forth in Table 13; Vi) the expression ofat least 2, 3, 4, 5, 6, 7, 8, 1,12,13,14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more ofthe rat ES cell-speciﬁc genes as set forth in Table 13; Vii) at least a 20-fold increase in the expression of one or more of the rat ES cell-speciﬁc genes as set forth in Table 13 when ed to a FlH4 mouse ES cell; Viii) at least a 20-fold increase in the sion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of the rat ES cell-specific genes as set forth in Table 13 when compared to a FlH4 mouse ES cell; ix) at least a 20-fold decrease in the sion of one or more of the rat ES cell-speciﬁc genes as set forth in Table 12 when compared to a FlH4 mouse ES cell; and/or x) at least a 20-fold decrease in the sion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of the rat ES cell-specific genes as set forth in Table 12 when compared to a FlH4 mouse ES cell; xi) any combination of expression of the rat ES cell-speciﬁc genes of parts (i)-(X); xii) a relative expression level of pluripotency markers as shown in Table for at least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17 or 18 ofthe listed otency markers. See, otency ranking column of Table 15 for relative expression levels; xiii) a relative expression level of the mesodermal markers as shown in Table 15 for at least 2, 3, or 4 of the listed mesodermal markers. See, mesodermal ranking column in Table 15 for relative expression levels; xiv) a relative expression level of endodermal markers as shown in Table for at least 2, 3, 4, 5, or 6 of the listed endodermal markers. See, endodermal ranking column in Table 15 for relative expression levels; xv) a relative expression level of neural markers as shown in Table 15 for at least 2 and 3 of the listed neural markers. See, neural ranking column in table 15 for relative expression levels; xvi) a relative expression level of trophectoderm s as shown in Table 15 for the listed trophectoderm markers. See, trophectoderm ranking column in table 15 for relative expression levels; xvii) any relative expression level of one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,2 4, 25, 26, 27, 28, 29 or 30) ofthe otency markers, mesodermal markers, rmal s, neural markers and/or trophectoderm markers set forth in Table 15; xviii) the relative expression level of each of the s set forth in table 15; xix) any combination of the signatures set forth in xii-xiix; and/or xx) any combination of the signature set forth in i-xiix; (j) have the y to produce a F0 rat; (k) capable of being subcultured and ining the undifferentiated state; (1) have the same number of chromosomes a normal rat cell; and/or (m) maintain pluripotency in vitro without ing paracrine LIF signaling. (11) have self renewal, meaning they divide indefinitely while maintaining pluripotency.

Such properties of a given rat ES cell line can be present at any one of the passage, including at passages 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, WO 30706 22, 23, 24, 25, 30, 40, 50 or later.

Thus, in one non-limiting embodiment, an in vitro culture sing a feeder cell layer and a population of rat ES cells is provided, wherein the in vitro culture conditions maintain pluripotency of the rat ES cells and comprises a media having mouse leukemia inhibitor factor (LIF) or an active variant or fragment thereof In specific embodiments, the rat ES cells cultured under such conditions maintains pluripotency of at least 50% of cell population over a period of at least 10 passages, maintains otency of at least 60% of cell population over a period of at least 10 passages, maintains pluripotency of at least 70% of cell population over a period of at least 10 passages, maintains pluripotency of at least 75% of cell population over a period of at least 10 passages, maintains pluripotency of at least 80% of cell population over a period of at least 10 passages, maintains pluripotency of at least 85% of cell tion over a period of at least 10 passages, ins pluripotency of at least 90% of cell population over a period of at least 10 passages, or maintains otency of at least 95% of cell tion over a period of at least 10 passages.

Further provided herein are in vitro cultures comprising the various rat ES cells, cell populations and cell lines disclosure herein, as well as, culturing kits for these various ES cells. For example, as sed above, in speciﬁc embodiments, the various rat ES cells provided herein have a one or more of the following characteristics: (1) when cultured in vitro loosely adhere to a feeder cell layer; (2) when cultured in vitro they form sphere-like colonies when plated on a feeder cell layer in vitro; (3) they maintain pluipotency when cultured in vitro under conditions comprising a feeder cell layer that is not genetically modified to express ia inhibitor factor (LIF), wherein the culture media comprises a sufﬁcient concentration of LIF; and/or (4) they capable of being subcultured and maintaining the undifferentiated state. Moreover, the rat ES cell populations of any of these in vitro cultures can comprise, for example, a population of cells in which at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells within the population are euploid, are diploid and/or have 42 chromosomes.

One method of culturing a rat nic stem cells in vitro ses (a) providing an in vitro culture comprising a feeder cell layer and a population of isolated rat embryonic stem (ES) cells; and (b) culturing the in vitro culture under ions which are sufficient to maintain pluipotency or totipotency of the isolated rat embryonic stem (ES) cell, and wherein the rat ES cells form colonies that loosely adhere to the feeder cell layer. “Loose adherence” or “adhere loosely” means that, if the culture dish is urbed for a period of time (minimum 8 hours), some colonies will adhere to the feeders such that they can maintain adherence if the dish is gently moved. In smaller wells (where the media moves less), loose adherence can happen faster. In either case, these colonies can be dislodged by either a) swirling the media in the dish or by gently pipetting media across the surface of the feeders. The morphology of these loosely adherent es is still spherical. In such instances the rat ES cells form sphere-like colonies when plated on a feeder cell layer in vitro. Such sphere-like colonies are shown for e in Figure 1. iv. Kits and In Vitro Cultures The rat ES cells and rat ES cell lines ed herein can be contained within a kit or an article of manufacture. In speciﬁc embodiments, the kit or article of cture comprises any of the rat ES cell lines or populations disclosed herein. The kit can further comprise any culture media that maintains the rat ES cell, including media that ins the pluripotency of the rat ES cells. Such media can comprise culture media having mouse LIF or an active variant or fragment thereof, as discussed in greater detail elsewhere herein. The media within the kit can ﬁarther comprise a MEK inhibitor and a GSK-3 inhibitor, or alternatively, the media within the kit can r comprise a combination of inhibitors consisting of a MEK inhibitor and a GSK-3 inhibitor. In speciﬁc embodiments, the media in the kit comprises a MEK inhibitor comprising PD032590l and/or a GSK-3 inhibitor comprising CHIR99021. Any of the various medias discussed herein, can be contained within the kit. r provided is a kit or article of manufacture that comprises any of the rat ES cell lines or populations disclosed herein, any of the various media disclosed , and a population of feeder cells. In one embodiment, the feeder cells in the kit or article of manufacture are not genetically modified to express LIF and/or the feeder cells comprises mitotically inactivated mouse embryonic ﬁbroblasts (MEFs). Any of the other feeder cells disclosed herein can be employed in the kit or article of manufacture.

IV. Genetic Modiﬁcation ofRat Embryonic Stem (ES) Cells The s rat ES cells and cell lines sed herein can be modiﬁed to contain at least one targeted genetic modiﬁcation. Thus, various methods are provided for genetically modifying an isolated rat embryonic stem (ES) cell as disclosed herein. The method comprises introducing into the genome of an ed rat ES cell disclosed herein a targeted genetic modiﬁcation to form a genetically modiﬁed rat ES cell. The targeted genetic modiﬁcation can comprise any modiﬁcation to the genome of rat ES including, for example, an insertion, a deletion, a knockout, a knockin, a mutation, or a combination thereof. In one embodiment, the targeted genetic ation comprises insertion of a heterologous cleotide into the genome of the rat ES cell. As used herein, “heterologous” in reference to a sequence is a ce that originates from a foreign species, or, if from the same species, is substantially modiﬁed from its native form in composition and/or genomic locus by deliberate human intervention.

In one aspect, an isolated rat ES cell or rat ES cell line is provided that is capable of ning pluripotency following one or more genetic modiﬁcations in vitro and that is capable of transmitting a genetically modiﬁed genome to a germline of an F1 generation. Thus, the rat ES cell maintains its pluripotency to develop into a plurality of cell types following the one or more serial genetic modiﬁcations in vitro (e.g., two, three, four, ﬁve, or six or more serial c modiﬁcations). In other embodiments, le targeted genetic modiﬁcations are made in a given rat ES cell, ing, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14, 15 or more. As such, multiple heterologous polynucleotides can also be integrated into the genome, including for example, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,1314,15 or more.

In one embodiment, following any one of the one to 15 serial genetic ations, the genetically modiﬁed rat ES cells upon exposure to differentiation medium are capable of differentiation into a plurality of cell types. In one embodiment, following any one of the one to 15 serial genetic modiﬁcations, the cally modiﬁed rat ES cells are capable of being maintained in an undifferentiated state in culture. In one embodiment, the genetically d and cultured rat ES cells in the undifferentiated state, when employed as donor cells in a rat host embryo, populate the embryo and form a blastocyst comprising the one to ﬁfteen genetic modiﬁcations. In one embodiment, the blastocyst, when implanted into a surrogate mother under conditions suitable for gestation, develops into an F0 rat progeny that comprises the one to 15 genetic modiﬁcations.

Various methods for making targeted genetic ations within a genome of a rat ES cell can be used. For example, in one instance, the targeted genetic modiﬁcation employs a system that will generate a ed genetic modiﬁcation via a homologous recombination event. In other instances, the rat ES cells can be modiﬁed using nuclease agents that generate a single or double strand break at a targeted genomic location. The single or double-strand break is then repaired by the non-homologous end joining pathway (NHEJ). Such s ﬁnd use, for example, in generating targeted loss of function genetic modifications. See, for example, Tesson et al. (2011) Nature Biotechnology 29:695-696, herein incorporated by reference. Such agents include, Transcription Activator-Like Effector Nuclease (TALEN) (; zer et al. (2010) PNAS 10.1073/pnas.1013133107; Scholze & Boch (2010) Virulence 1:428- 432; Christian et al. Genetics (2010) 186:757-761; Li et al. (2010) Nuc. Acids Res. (2010) doi:10.1093/nar/gkq704; and Miller et al. (2011) Nature Biotechnology 29: 143— 148; US Patent ation No. 2011/0239315 A1, 2011/0269234 A1, 2011/0145940 A1, 2003/0232410 A1, 2005/0208489 A1, 2005/0026157 A1, 2005/0064474 A1, 2006/0188987 A1, and 2006/0063231 A1 (each hereby incorporated by reference); a zinc-ﬁnger nuclease (ZFN) (US20060246567; US20080182332; US20020081614; US20030021776; WO/2002/057308A2; US20130123484; US20100291048; and, WO/2011/017293A2, each ofwhich is herein orated by reference); a meganuclease (see, Epinat et al., (2003) Nucleic Acids Res 31 62; Chevalier et al., (2002) Mol Cell -905; Gimble et al., (2003) Mol Biol 334:993-1008; Seligman et al., (2002) Nucleic Acids Res 0-9; Sussman et al., (2004) JMol Biol 342:31-41; Rosen et al., (2006) Nucleic Acids Res 34:4791-800; Chames et al., (2005) Nucleic Acids Res 33:e178; Smith et al., (2006) Nucleic Acids Res 34:e149; Gruen et al., (2002) Nucleic Acids Res 30:e29; Chen and Zhao, (2005) Nucleic Acids Res 33:e154; WO2005105989; WO2003078619; WO2006097854; WO2006097853; WO2006097784; and WO2004031346); and a CAS/CRISPER system (Mali P et al. (2013) Science 2013 Feb 15;339(6121):823-6; Jinek M et al. e 2012 Aug 17;337(6096):816-21; Hwang WY et al. Nat Biotechnol 2013 Mar;31(3):227-9; Jiang W et al. Nat Biotechnol 2013 Mar;31(3):233-9; and, Cong L et al. Science 2013 Feb 15;339(6121):819-23, each of which is herein incorporated by reference).

In other ments, the targeted genomic modiﬁcation can be made by employing a homologous recombination targeting vector. In such instances, the targeting vector ses the insert polynucleotide and ﬁarther comprises an am and a ream homology arm which ﬂank the insert polynucleotide. The homology arms which ﬂank the insert cleotide correspond to genomic regions within the ed genomic locus. For ease of reference, the corresponding genomic regions within the targeted genomic locus are referred to herein as “target sites”. Thus, in one example, WO 30706 a targeting vector can comprise a ﬁrst insert polynucleotide ﬂanked by a ﬁrst and a second homology arm corresponding to a ﬁrst and a second target site located at the targeted genomic locus. The targeting vector y aids in the integration of the insert polynucleotide into the targeted genomic locus through a homologous recombination event that occurs between the homology arms and the ponding target sites within the genome of the cell.

As used , a homology arm and a target site spond” or are “corresponding” to one another when the two regions share a sufﬁcient level of sequence identity to one another to act as substrates for a homologous recombination reaction. By “homology” is meant DNA sequences that are either identical or share sequence identity to a ponding sequence. The sequence identity between a given target site and the ponding homology arm found on the targeting vector can be any degree of sequence identity that allows for homologous recombination to occur. For example, the amount of sequence identity shared by the homology arm of the targeting vector (or a fragment f) and the target site (or a fragment thereof) can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% sequence identity, such that the sequences undergo homologous recombination. Moreover, a corresponding region of homology between the homology arm and the corresponding target site can be of any length that is sufﬁcient to promote homologous recombination at the cleaved recognition site.

In speciﬁc embodiments, the isolated rat ES cell, cell line or cell population exhibits a homologous recombination efﬁciency of at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 25%, at least %, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%. In one ment, the homologous recombination efﬁciency employing the rat ES cell is greater than 4%.

In speciﬁc embodiments, a selection marker is employed when generating a targeted genetic modiﬁcation in a rat ES cell. The polynucleotide encoding the selection marker can be present on the ing vector which is designed to uce the targeted genetic modiﬁcation into the genome, or it can be found on a separate plasmid or vector.

The polynucleotide ng the selection marker can be contained within in expression cassette. The various components of such expression cassettes are discussed in r detail elsewhere herein.

Various selection markers can be used in the methods and compositions disclosed .

Such selection markers can, for example, impart resistance to an antibiotic such as G418, hygromycin, blastocidin, puromycin or neomycin. Such ion markers include neomycin phosphotransferase (neor), hygromycin B phosphotransferase (hygr), puromycin N—acetyl-transferase and cidin S deaminase (bsrr). In still other embodiments, the selection marker is operably linked to an inducible promoter and the expression of the selection marker is toxic to the cell. miting examples of such selection markers include xanthine/guanine phosphoribosyl erase (gpt), hahypoxanthine-guanine phosphoribosyltransferase (HGPRT) or herpes simplex virus thymidine kinase K). See, for example, Santerre et al. (1984) Gene 30:147-56; Joyner (1999) The Practical Approach Series, 293; Santerre et al. (1984) Gene 30: 147- 56; Bernard et al. (1985) Exp Cell Res 158:237-43; Giordano and McAllister (1990) Gene, 88:285-8; Izumi et al. (1991) Exp Cell Res 197:229-33), each ofwhich is herein incorporated by reference in their entirety. In speciﬁc embodiments, the neoR able marker is the neomycin phosphotransferase (ne0) gene of Beck et al. (1982) Gene, 19:327-36, which is herein incorporated by reference. The neoR selection maker is that used in US Patent No, 7,205,148 or 6,596,541, each of which are herein incorporated by reference.

In speciﬁc embodiments, the selection marker employed is a non-attenuated ion marker. A “non-attenuated selection marker” comprises a selection marker that retains the ty of the native polypeptide or the selection marker has an increased activity when compared to the native form of the ptide. An increased in activity of a ion marker can comprise any statistically signiﬁcant increase in activity including, for example, an increase of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% 80% or higher. Thus, a non-attenuated ion marker when expressed in a host cell will allow for a higher percentage of the host cells to survive in the presence of a higher concentration of the selection agent than when employing an attenuated selection marker. Non-attenuated selection markers include, for example, a neomycin non- attenuate selection marker. See, for e, Beck et al. (1982) Gene, 19:327-36 or in US Patent No, 7,205,148 or 6,596,541, each of which are herein incorporated by reference.

In other ces, the sed activity of a selection marker when compared to an attenuated selection marker and/or the wild type (native) selection marker can result from increasing the copy number of either the non-attenuated, attenuated or native selection marker within the genome of the rat ES cell. As such, a given rat ES cell can comprise within its genome at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of a given selection marker (i.e., a non-attenuated selection marker, an attenuated selection marker or a native (wildtype) selection marker).

The various selection markers ed are encoded by a polynucleotide operably linked to a promoter active in the rat ES cell. In speciﬁc embodiments, the increase in the activity of the ion marker can result from an increase in expression of the selection marker. Thus, a er can be employed to elevate the expression levels of a given ion marker. Promoters of interest e, but are not limited to, the CMV er, the PGK promoter and the CAG promoter. In one embodiment, the human Ubiquitin (hUb) promoter is used to express the selection marker. See, Valenzuela et al. (2003) Nature hnology 21:652-659, herein incorporate by reference in its entirety.

In one embodiment, the rat ES cell ins its otency to develop into a plurality of cell types following a single round of electroporation with an exogenous nucleic acid. In r embodiment, the rat ES cell maintains its pluripotency to develop into a plurality of cell types following a second round of oporation with an exogenous nucleic acid, following a third round of electroporation with an exogenous nucleic acid, following a fourth round of electroporation with an exogenous nucleic acid, following a ﬁfth round of electroporation with an exogenous nucleic acid, following a sixth round of electroporation with an exogenous nucleic acid, following a seventh round of electroporation with an ous nucleic acid, ing an eighth round of electroporation with an exogenous nucleic acid, following a ninth round of electroporation with an exogenous nucleic acid, following a tenth round of electroporation with an exogenous nucleic acid, following an eleventh round of electroporation with an exogenous nucleic acid, following a twelfth round of electroporation with an exogenous nucleic acid, following a thirteenth round of electroporation with an exogenous nucleic acid, following a fourteenth round of electroporation with an exogenous nucleic acid and/or following a ﬁfteenth round of electroporation with an exogenous nucleic acid. In other embodiments, the rat ES cell is capable of transmitting a targeted genetic modiﬁcation into progeny following a 2014/017452 successive round of electroporation (i.e., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds of electroporation). 1'. Introducing Sequences into a Rat Embryonic Stem Cell The methods provided herein comprise ucing into a cell with one or more polynucleotides or polypeptide constructs comprising the various components needed to make the targeted genomic modification. "Introducing" means presenting to the cell the sequence (polypeptide or polynucleotide) in such a manner that the sequence gains access to the interior of the cell. The methods ed herein do not depend on a particular method for introducing any component of the targeted genomic integration system into the cell, only that the polynucleotide gains access to the interior of a least one cell. s for introducing polynucleotides into various cell types are known in the art and include, but are not limited to, stable transfection methods, transient transfection methods, and virus-mediated s. Such method include, but are not limited to, electroporation, ytoplasmic injection, viral infection (including adenovirus, lentivirus, and retrovirus vectors), transfection, lipid-mediated ection and/or factionTM. See, for example, Stadtfeld et al. (2009) Nature Methods 6(5):329-330; Yusa et al. (2009) Nat. Methods 6:363-369; Woltjen et al. (2009) Nature 458, 766-770. Such methods include, but are not limited to, direct delivery ofDNA such as by ex vivo transfection (Wilson et al., Science, 244: 1344-1346, 1989, Nabel and Baltimore, Nature 326:71 1-713, 1987), optionally with Fugene6 (Roche) or Lipofectamine (Invitrogen), by injection (US. Pat. Nos. 5,994,624, 5,981,274, ,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including nj ection (Harland and Weintraub, J.

Cell Biol., 101 :1094-1099, 1985; US. Pat. No. 5,789,215, incorporated herein by reference); by electroporation (US. Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter et al., Proc. Nat'l Acad. Sci.

USA, 81 :7161-7165, 1984); by calcium ate precipitation (Graham and Van Der Eb, Virology, -467, 1973; Chen and Okayama, Mol. Cell Biol., 745-2752, 1987; Rippe et al., Mol. Cell Biol., 10:689-695, 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, Mol. Cell Biol., 5:1188-1190, 1985); by direct sonic loading (Fechheimer et al., Proc. Nat'l Acad. Sci. USA, 84:8463-8467, 1987); by me mediated ection (Nicolau and Sene, Biochim. Biophys. Acta, 721 :185- 190, 1982; Fraley et al., Proc. Nat'l Acad. Sci. USA, 76:3348-3352, 1979; Nicolau et al., Methods Enzymol., 149:157-176, 1987; Wong et al., Gene, 10:87-94, 1980; Kaneda et al., Science, 243:375-378, 1989; Kato et al., Biol. Chem, 266:3361-3364, 1991) and receptor-mediated transfection (Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987); and any combination of such methods, each of which is incorporated herein by reference.

In some ments, the cells employed in the methods and compositions have a DNA construct stably incorporated into their genome. "Stably incorporated" or y uced" means the introduction of a polynucleotide into the cell such that the nucleotide sequence integrates into the genome of the cell and is capable of being inherited by progeny thereof. Any protocol may be used for the stable incorporation of the DNA constructs or the various components employed to generate the targeted genomic ation.

Transfection protocols as well as protocols for introducing polypeptides or polynucleotide sequences into cells may vary. miting transfection methods include chemical-based transfection methods include the use of mes; rticles; calcium phosphate (Graham et al. (1973). Virology 52 (2): , Bacchetti et al. (1977) Proc Natl Acad Sci USA 74 (4): 1590—4 and, Kriegler, M (1991). Transfer and Expression: A Laboratory Manual. New York: W. H. Freeman and Company. pp. 96— 97); dendrimers; or cationic rs such as DEAE-dextran or polyethylenimine. Non chemical s include electroporation; Sono-poration; and optical transfection .

Particle-based transfection include the use of a gene gun, magnet assisted ection (Bertram, J. (2006) Current Pharmaceutical Biotechnology 7, 277—28). Viral methods can also be used for transfection.

A non-limiting example, of a method of introduction a heterologous polynucleotide into a rat ES cell follows. The rat ES cells, as described herein, are passaged for about 24 to about 48 prior to electroporation. About 24 hours prior to electroporation the media is changed to RVG2i + ROCKi (10uM 2). The rat ES cells are trypsinized and the rat ES cells are isolated. The rat ES cells are suspended to achieve a final cell concentration of about 2 x 10A6 to about 10 X 10A6 cells per 75 ul.

About 752 of rat ES cells are added to about 507t DNA comprising the heterologous polynucleotide and about 125% EP buffer is added. In one non-limiting ment, the electroporation is carried out with the following parameters: 400V; 400V; 9; 100 uF.

The cells are then cultured in RVG2i and 10uM ROCKi and can be transferred onto feeder cells. ii. Selecting Rat Embryonic Stem Cells Having a Targeted Genomic Modiﬁcation s method are provided for selecting and maintaining rat ES cells having stably incorporated into their genome a targeted genetic modiﬁcation. In one non- limiting example, when introducing a heterologous polynucleotide into a rat ES cell, the method can comprise (a) providing an in vitro population of rat ES cells; (b) introducing into at least one rat ES cell a heterologous polynucleotide comprising a selection marker operably linked to a promoter active the rat ES cell; and, (c) culturing in vitro the rat ES cell population in an alternating ﬁrst and second culture media wherein the ﬁrst culture media comprises an effective amount of a selection agent for a ﬁrst time period and the second culture media do not comprise the selection agent, wherein the in vitro culture conditions in pluripotency or totipotency; and thereby selecting the rat ES cell having stably integrated into its genome the heterologous polynucleotide. The s methods by which the rat ES cell having the targeted genetic modiﬁcation can be selected in a given population can employ an in vitro culture system which allows the rat ES cells to maintain pluripotency. Thus, any of the in vitro culture medias and feeder cells discussed herein can be employed.

In speciﬁc embodiments, the ﬁrst and the second culture media are ated about every 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, , 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 hours or more. In a speciﬁc embodiment, the ﬁrst and second culture media are alternated every 24 hours.

Any appropriate selection marker can be used and the corresponding selection agent will be present at an ive tration with the culture media. Such selection markers include any of the native, attenuated or non-attenuated selection marker discussed herein. In one embodiment, the ion marker employed s resistance to an antibiotic, including for example, G418. miting selection s comprise in phosphotransferase II (nptII) or hygromycin phosphotransferase (hpt).

The concentration of the selection agent is such as to allow for the selection of a rat ES cell having the selection marker while maintaining the pluipotency of the rat ES cells that are present within the culture. When employing, for example, G418, the concentration of the G418 in the selection media can range from about 50 11ng to about 125 ug/ml, about 60 ug/ml to about 125ug/ml, about 70 11ng to about ml, about 80ug/ml to about 125ug/ml, about 90ug/ml to about 125ug/ml, about 100ug/ml to about 125 ug/ml, about 110 ug/ml to about 125ug/ml, about 80ug/ml to about 100ug/ml, about l to about 85ug/ml, about 70ug/ml to about 80ug/ml. In one embodiment, the concentration of G418 in the culture is 75 ug/ml.

The media employed in the selection allows the rat nic stem cells to retain pluripotency. Such media is described in detail elsewhere herein.

The selection protocol can be initiated at any time following the introduction of the polynucleotide encoding the selection marker into the genome of the rat ES cell. In speciﬁc embodiments, the selection protocol begins 10, 15, 20, 24, 30, 35, 40, 50, 60 or more hours after the introduction of the selection marker into the rat ES cell. In one embodiment, the selection protocol beings about 2 days following the introduction of the polynucleotide encoding the ion marker.

A non-limiting selection protocol employing G418 is as follows. Day 2, (211d day after the introduction of the polynucleotide encoding the selection marker) the population of rat ES cells is incubated cells in 2i media and G418 at 75 ug/ml. At day 3, the tion of rat ES cells are incubated cells in 2i media without G418. At day 4, the population of rat ES cells are incubated in 2i media and G418 at 75 ug/ml. At day 5, the population of rat ES cells are incubated cells in 2i media t G418. At day 6, the tion of rat ES cells are ted in 2i media and G418 at 75 ug/ml. At day 7, the population of rat ES cells is incubated in 2i media without G418. At day 8, the population of rat ES cells is incubated in in 2i media without G418 and 75 ug/ml. At day 9, the population of rat ES cells is incubated cells in 2i media without G418. At day 10, the population of rat ES cells is incubated cells in 2i media and G418 at 75 ug/ml. At day 11, the population of rat ES cells is incubated cells in 2i media without G418. At day 12, colonies are picked for expansion and screening.

Following the selection of the rat ES cells haVing the selection marker, the colonies can be expanded. In specific embodiments, the period for expansion can be about 1, 2, 3, 4, 5, or more days in a e condition that maintains the pluripotency of the cells. In one miting embodiment, the selected colonies are expanded for 3 days. In a further ment, the media employed is a 2i media. Each clone can then be passed and ﬁarther expanded.

WO 30706 The rat ES cells and cell lines having one or more of the targeted c modiﬁcations can have one or more any the following properties: (a) have germ-line competency following targeted genetic modiﬁcation, meaning when the rat ES cell is implanted into a rat host , the targeted genetic modiﬁcation within the genome of the rat ES cell line, is transmitted into an offspring; (b) have pluripotency in vitro; (c) have totipotency in vitro; (d) when cultured in vitro loosely adhere to a feeder cell layer; (e) when cultured in vitro form sphere-like es when plated on a feeder cell layer in vitro; (f) maintain pluripotency when cultured in vitro under conditions comprising a feeder cell layer that is not genetically modiﬁed to express leukemia inhibitor factor (LIF), wherein the culture media comprises a sufﬁcient concentration of LIF; (g) maintain pluripotency when cultured in vitro under conditions comprising a feeder cell layer, wherein the culture media ses mouse LIF or an active variant or fragment thereof; (h) comprise a molecular signature characterized by i) the expression of one or more of rat ES cell-speciﬁc genes sing Adheres Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-Al (Efnal), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gij), Insulin-like growth factor binding protein-like l (Igfbpll), Interleulin 36 beta(Illf8), Interleukin 28 receptor, alpha (Il28ra), left-right determination factor 1 (Leftyl), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid or 2 (Lpar2), Neuronal pentraxin or (Ntm), Protein tyrosine phosphatase non-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III and n repeat domains 1 (Fankl), Forkhead box El (thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box El (thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lefl ), Sal-like 3 (Drosophila) ), SATB ox l (Satbl), miR-632, or a combination thereof; ii) the expression ofat least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,l3,l4,15,l6, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more of the rat ES cell-speciﬁc genes comprising s Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-Al (Efna1), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gjb5), Insulin-like growth factor binding protein-like 1 (Igfbpll), Interleulin 36 beta(111f8), Interleukin 28 receptor, alpha (Il28ra), left-right ination factor 1 (Leftyl), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III and ankyrin repeat domains 1 (Fankl), Forkhead box E1 (thyroid transcription factor 2) ), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid transcription factor 2) (Foxel), enhancer-of-split related with YRPW motif 2 , Lymphoid enhancer-binding factor 1 (Lefl ), Sal-like 3 (Drosophila) (Sall3), SATB homeobox 1 (Satbl), miR-632, or a combination thereof; iii) at least a 20-fold increase in the expression of one or more of the rat ES cell-speciﬁc genes as set forth in Table 14 when ed to a F1H4 mouse ES cell; iV) at least a d increase in the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more ofthe rat ES peciﬁc genes as set forth in Table 14 when compared to a F1H4 mouse ES cell; V) the expression of one or more of rat ES cell-speciﬁc genes as set forth in Table 13; Vi) the expression ofat least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more ofthe rat ES cell-speciﬁc genes as set forth in Table 13; Vii) at least a 20-fold increase in the expression of one or more of the rat ES cell-speciﬁc genes as set forth in Table 13 when compared to a F1H4 mouse ES cell; Viii) at least a 20-fold increase in the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of the rat ES peciﬁc genes as set forth in Table 13 when compared to a F1H4 mouse ES cell; ix) at least a 20-fold decrease in the expression of one or more of the rat ES cell-speciﬁc genes as set forth in Table 12 when compared to a F1H4 mouse ES cell; and/or x) at least a 20-fold decrease in the expression of at least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17,18,19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or WO 30706 more of the rat ES pecific genes as set forth in Table 12 when compared to a F1H4 mouse ES cell; xi) any combination of expression of the rat ES cell-speciﬁc genes of parts (i)-(X); xii) a relative expression level of pluripotency markers as shown in Table for at least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17 or 18 ofthe listed pluripotency markers. See, pluripotency ranking column of Table 15 for relative expression levels; xiii) a relative expression level of the mesodermal markers as shown in Table 15 for at least 2, 3, or 4 of the listed mesodermal markers. See, rmal ranking column in Table 15 for relative expression levels; xiv) a relative sion level of rmal markers as shown in Table for at least 2, 3, 4, 5, or 6 of the listed endodermal s. See, endodermal ranking column in Table 15 for relative expression levels; xv) a relative expression level of neural markers as shown in Table 15 for at least 2 and 3 of the listed neural markers. See, neural ranking column in table 15 for relative expression levels; xvi) a relative expression level of trophectoderm markers as shown in Table 15 for the listed trophectoderm markers. See, trophectoderm ranking column in table 15 for ve expression levels; xvii) any relative expression level of one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,2 4, 25, 26, 27, 28, 29 or 30) ofthe pluripotency markers, mesodermal s, endodermal markers, neural markers and/or trophectoderm markers set forth in Table 15; xviii) the relative expression level of each of the markers set forth in table 15; xix) any combination of the signatures set forth in xii-xiix; and/or xx) any combination of the signature set forth in i-xiix. (i) have the y to produce a F0 rat; (j ) capable of being subcultured and maintaining the undifferentiated state; (k) having the same number of chromosomes a normal rat cell; (I) maintain pluripotency in vitro without requiring paracrine LIF signaling; and/or WO 30706 2014/017452 (m) have self renewal, meaning they divide indeﬁnitely while ining pluripotency. z'l'l'. Expression Cassettes The terms “polynucleotide,3, ECpolynucleotide sequence, 3) CEnucleic acid ce,” and “nucleic acid fragment” are used hangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer ofDNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Polynucleotides can se deoxyribonucleotides and cleotides include both naturally occurring molecules and synthetic analogues, and any combination these. The polynucleotides provided herein also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

Further provided are recombinant polynucleotides. The terms “recombinant polynucleotide” and “recombinant DNA construct” are used interchangeably herein. A recombinant construct comprises an artiﬁcial or heterologous combination of nucleic acid sequences, e.g., regulatory and coding sequences that are not found together in nature. In other embodiments, a recombinant construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in . Such a construct may be used by itself or may be used in conjunction with a vector. If a vector is used, then the choice of vector is dependent upon the method that is used to transform the host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. Genetic ts required to successfully transform, select and propagate host cells and comprising any of the isolated nucleic acid fragments provided herein. Screening may be accomplished by Southern analysis of DNA, Northern analysis ofmRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, among others.

In speciﬁc embodiments, one or more of the components described herein can be provided in an expression te for expression in a rat cell. The cassette can include 5' and 3' regulatory sequences ly linked to a polynucleotide ed herein.

“Operably ” means a ﬁanctional linkage between two or more elements. For e, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a er) is a ﬁanctional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non- contiguous. When used to refer to the g of two protein coding regions, operably linked means that the coding regions are in the same reading frame. In r instance, a nucleic acid sequence encoding a protein may be operably linked to regulatory sequences (e.g., promoter, enhancer, silencer sequence, etc.) so as to retain proper riptional regulation. The cassette may additionally n at least one additional polynucleotide of st to be co-introduced into the rat ES cell. Alternatively, the onal polynucleotide of interest can be provided on multiple expression cassettes.

Such an sion cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of a recombinant polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression te may additionally contain selection marker genes.

The expression cassette can include in the 5'-3' direction of transcription, a transcriptional and ational initiation region (i.e., a promoter), a recombinant polynucleotide provided , and a transcriptional and translational termination region (i.e., termination region) functional in mammalian cell or a host cell of interest. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or a polynucleotide provided herein may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or a polynucleotide ed herein may be heterologous to the host cell or to each other.

For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the s from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or c locus, or the promoter is not the native promoter for the operably linked polynucleotide. Alternatively, the regulatory regions and/or a recombinant polynucleotide provided herein may be entirely synthetic.

The termination region may be native with the transcriptional initiation region, may be native with the ly linked recombinant polynucleotide, may be native with the host cell, or may be derived from another source (i.e., foreign or heterologous) to the promoter, the recombinant polynucleotide, the host cell, or any combination thereof In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to e for convenient restriction sites, removal of superﬂuous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e. g., transitions and transversions, may be involved.

A number of promoters can be used in the expression cassettes provided herein.

The promoters can be selected based on the desired outcome. It is recognized that different applications can be enhanced by the use of ent promoters in the expression cassettes to te the timing, location and/or level of expression of the polynucleotide of st. Such expression constructs may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible, constitutive, environmentally- or developmentally-regulated, or cell- or tissue-speciﬁc/selective expression), a transcription tion start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal. iv. Generating F0 Rat Embryos and F1 Progeny Having the Targeted Genetic ation The various methods and compositions provided herein can be used to generate a genetically modiﬁed rat. Such methods generally comprise (a) introducing into the genome of an ed rat ES cell disclosed herein a targeted genetic modiﬁcation to form a rat ES cell having a genetic modiﬁcation; (b) implanting at least one of the cally modiﬁed rat ES cells having the genetic modiﬁcation into a rat host embryo to produce a F0 embryo; (c) implanting the F0 embryo into a surrogate mother; (d) gestating the F0 embryo in the surrogate mother to term; and, (e) identifying a F0 rat having the c modiﬁcation.

The genetically modiﬁed rat ES cells having the genetic modiﬁcation can be ted into a rat host embryo that is from the same rat strain or from a different rat strain. For example, a genetically modiﬁed DA rat ES cell can be implanted into a DA rat host embryo or it can be implanted into an SD host , ACI host embryo or other heterologous rat host embryo. Similarly, a genetically modiﬁed ACI rat ES cell can be introduced into an ACI rat host embryo or it can be introduced into an SD host , DA host embryo or other heterologous rat host . Likewise, the surrogate mother can be from the same rat strain as the genetically modiﬁed rat cell and/or the rat host embryo or the surrogate mother can be from a different rat strain as the genetically modiﬁed rat cell and/or the rat host embryo. In one non-limiting embodiment, the genetically modiﬁed rat cell is from a DA strain, the host rat embryo is from an SD host embryo and the surrogate mother is from a DA strain. In another non-limiting embodiment, the cally modiﬁed rat cell is from an ACI strain, the host rat embryo is from an SD strain, and the surrogate mother is from a DA strain.

In still r embodiments, the chimeric rat (F0) can be breed to produce an F1 y that is zygous for the targeted genetic ation. In addition, the male rat of the F1 progeny can be breed with a female rat of the Fl progeny to obtain an F2 progeny that is homozygous for the genetic modiﬁcation.

The methods and compositions provided herein allow for at least 1%, 3%, 5%, %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or greater of the F0 F0 rats having the genetic modiﬁcation to transmit the genetic modiﬁcation to the F1 progeny.

In some embodiments, the rat ES cells having the targeted genetic modiﬁcation are introduced into a pre-morula stage embryo from a corresponding sm, e.g., an 8- cell stage mouse embryo. See, e.g., US 7,576,259, US 7,659,442, US 7,294,754, and US 2008-0078000 A1, all of which are incorporated by nce herein in their entireties. In other embodiments, the For, the donor rat ES cells may be implanted at 4 cell stage, 8 cell stage of the host embryo.

The rat embryos comprising the genetically modiﬁed rat ES cells is incubated until the blastocyst stage and then implanted into a surrogate mother to produce an F0.

Rats bearing the genetically modiﬁed genomic locus can be identiﬁed via modiﬁcation of allele (MOA) assay as described herein. The resulting F0 generation derived from the genetically modiﬁed rat ES cells is crossed to a wild-type rat to obtain F1 generation offspring. Following genotyping with speciﬁc primers and/or probes, F1 rats that are heterozygous for the genetically modiﬁed genomic locus are crossed to each other to produce a rat that is homozygous for the genetically modiﬁed genomic locus. r provided is a F0 rat embryo comprising an inner cell mass having at least one heterologous stem cell comprising any one of the rat ES cells ed herein. In other embodiments, progeny of a rat F0 embryo are provided n at least 50%, 60%, 70% or more of the F0 progeny are derived from a genetically modiﬁed rat ES cell of as sed herein.

In one aspect, a method for making a rat ES cell is ed, comprising ng from a morula-stage rat embryo, a blastocyst-stage rat embryo, or a rat embryo at a developmental stage between a morula-stage embryo and a blastocyst-stage embryo a rat cell, and ing the rat cell from the rat embryo under conditions sufﬁcient to maintain pluripotency and/or totipotency. In on ment, the conditions sufﬁcient to maintain pluripotency and/or totipotency include 2i media.

In one aspect, a method for making a genetically modiﬁed rat is ed, comprising a step of modifying a rat ES cell genome with a nucleic acid sequence of interest to form a modiﬁed rat ES cell, and employing the modiﬁed rat ES cell as a donor rat ES cell, combining the rat donor ES cell with a rat host embryo, ing the donor ES cell and rat host embryo, and employing the cultured host embryo to make a genetically modiﬁed rat.

In one , a method for making genetically modiﬁed rat Fl progeny is ed, comprising a step of modifying a rat ES cell genome with a nucleic acid sequence of interest to form a modiﬁed rat ES cell, and employing the modiﬁed rat ES cell as a donor rat ES cell, combining the rat donor ES cell with a rat host embryo, culturing the donor ES cell and rat host embryo, and employing the cultured host embryo to make a genetically modiﬁed rat, wherein the progeny are about 3%, about 10% or more, or about 63% or more derived from the genetically modiﬁed donor rat ES cell.

In one embodiment, the cultured host embryo is implanted into a surrogate rat mother, and the cultured host embryo is gestated in the surrogate mother.

In one aspect, a method of transmitting a genetic modiﬁcation from a rat pluripotent cell to a rat progeny with high frequency is ed, comprising genetically modifying a pluripotent rat cell with a nucleic acid sequence of interest on a bacterial artiﬁcial chromosome to form a genetically modiﬁed rat pluripotent cell, and employing the genetically modiﬁed rat pluripotent cell with a rat host embryo in a rat surrogate mother to te a progeny comprising the genetic modiﬁcation and, optionally, breeding the progeny.

In one , a method for making a rat ES cell is provided, wherein the method comprises culturing a frozen 8-cell stage embryo to a blastocyst stage, and deriving from the cultured blastocyst a rat cell, and culturing the rat cell under conditions sufﬁcient to maintain otency and/or tency.

V. Variants, Fragments and ce Identity Active variants and fragments of the disclosed LIF polypeptide, particularly the mouse LIF polypeptide are provided . “Variants” refer to substantially similar sequences. As used , a "variant polypeptide” is intended to mean a polypeptide derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N—terminal and/or C-terminal end of the native protein; deletion and/or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant polypeptides continue to possess the desired biological ty of the native polypeptide, that is, they inhibit the differentiation of rat and/or mouse embryonic stem cells and contribute to stem cell self-renewal. A variant of a polypeptide or disclosed herein (i.e.

SEQ ID NOS: 1 or rot Accession No. ) will typically have at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the reference sequence.

The term “fragment” refers to a portion of an amino acid comprising a speciﬁed number of contiguous amino acid. In ular embodiments, a fragment of a polypeptide disclosed herein may retain the biological activity of the full-length polypeptide and hence inhibit the entiation of rat and/or mouse embryonic stem cells and contribute to stem cell self-renewal. Fragments of a polypeptide sequence disclosed herein (i.e. SEQ ID NOS: 1 or SwissProt Accession No. P09056) may se at least 10, 15, 25, 30, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, contiguous amino acids, or up to the total number of amino acids present in a full-length n.

As used herein, "sequence identity" or "identity" in the context of two polynucleotides or ptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of ce identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.

When sequences differ in conservative tutions, the percent ce identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full ch, thereby sing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score n zero and 1.

The scoring of conservative tutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).

As used herein, "percentage of sequence ty" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may se ons or deletions (i.e., gaps) as ed to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of d positions, ng the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

Unless otherwise , sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. "Equivalent m" means any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical t sequence identity when compared to the corresponding alignment generated by GAP n 10.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES Example 1: Rat ES Cell Derivation. rESC Characterization. As shown in rESC grow as compact spherical colonies which routinely detach and float in the dish (close-up, . . B, C, F, G: rESC express pluripotency markers including Oct-4 () and Sox2 (), and express high levels of alkaline phosphatase ( left . Karyotype for line DA.2B is 42X,Y( right panel). rESC often become tetraploid; thus, lines were pre-screened by ng metaphase some spreads; lines with mostly normal counts were then formally karyotyped.

ACI blastocysts were collected from super-ovulated females obtained commercially; DA blasts were cultured from frozen 8-cell embryos obtained commercially. Zona idae were removed with Acid s and blasts were plated onto mitotically inactivated MEFs. Outgrowths were picked and expanded using standard methods. All blasts were plated, cultured and expanded using 2i media (Li et al. (2008) Germline competent embryonic stem cells derived from rat blastocysts, Cell 135: 1299-13 10; incorporated herein by reference).

Table 3. Rat ES Cell Derivation Embryo source Blastocysts Frozen 8-cell embryos cultured to (Superovulation) blastocyst Outgrowths: 32 (30% of blasts) 10 (45% of blasts) Lines: 16 (50% of 9 (90% of outgrowths) wths) Karyotyped: 3; all 42X,Y 6: 3 42X,X 3 42X,Y GLT validated: l l) l 42X,X (DA.2C) 1 42X,Y (DA.2B) Example 2: Rat Production Chimeric rats were produced by blastocyst injection and transmission of the rESC genome through the germline. Chimeras produced by blastocyst microinjection using parental ACI.Gl rESC are shown in Fl agouti pups with albino littermates, sired by the ACI/SD chimera labeled with an asterisk (*) in are shown in ne transmission of parental rESC. Three euploid rESC lines were evaluated for pluripotency by microinjection into albino SD blastocysts. Chimeras were identified by agouti coat color indicating rESC contribution. For each line, a majority of chimeras transmitted the rESC genome to F1 offspring (Table 4).

Table 4. Germline Transmission of Parental rESC Total pups rESC- GLT Chimeras Germline Line from GLT derived efficiency bred transmitters chimeras pups (%) ACI.G1 5 3 (60%) 103 11 11 DA.2B 5 4 (80%) 129 11 9 DA.2C 3 2 (66%) 45 7 16 (XX) Example 3: rESC Targeting: The Rat Rosa 26 Locus.

The rRosa26 locus lies between the Setd5 and Thumpd3 genes as in mouse, with the same spacing. The rRosa 26 locus ( Panel B) differs from the mRosa 26 locus ( Panel A). The mRosa26 transcripts consist of 2 or 3 exons. The rat locus contains a 2nd exon 1 (Ex1b) in on to the homologous exon to mouse exon1 (Ex1a). No 3rd exon has been identified in rat. Targeting of a rRosa26 allele is depicted in Panel C, where homology arms of 5kb each were cloned by PCR using genomic DNA from DA rESC. The targeted allele ns a Z-hUb-neo cassette ing a 117bp deletion in the rRosa26 intron.

Targeting efficiency at the rRosa 26 locus was determined (Table 5). Linearized vector was electroporated into DA or ACI rESC, and ected colonies were cultured in 2i media + G418, using standard techniques. Individual colonies were picked and screened using a Loss of Allele (LOA) assay (Valenzuela, D. et al. (2003) High- throughput engineering of the mouse genome coupled with high-resolution expression is, Nature Biotech. 21 60, incorporated herein by reference).

Table 5. rRosa26 Targeting Efficiency Colonies Reconfirmed Targeting efficiency Cell line picked. . . pos1t1ves (%) ACI.Gl Chimera production and germline transmission using targeted Rosa26 rESC.

Reconfirmed targeted rRosa26 clones were microinjected into SD cysts, which were then transferred to pseudopregnant DS recipient females, using standard techniques. Chimeras were identified by coat color; male F0 chimeras were bred to SD females. ne (agouti) Fl pups were genotyped for the presence of the targeted Rosa26 allele; nine of 22 agouti pups genotyped as heterozygous at the Rosa26 locus (Table 6).

Table 6. Germline Transmission Using Targeted Rosa26 rESC R26 Clones Germline rESC- dErSiEe-d Cell line clones producing Transmitting derived . . . pups 1n] ected Ch1meras Clones Pups ((y )0 AH7: 64 AH7: 4l AH7: 63 AE3: ll2 AE3: 6 AE3: 3 DE9: 4 10 Exam le 3: Derivation of Rat Embr onic Stem Cells.

Superovulation protocol, rats Day 0: ed with pregnant mare serum: IP, 20 U (0.4 ml).

Day 1 : no action 2014/017452 Day 2: (46 hr. later): injected with hCG, IP, 50 U (1 ml). - set up single female matings.

Day 3: checked plugs. Females were d. This is day 0.5.

Day 6 (e35): Euthanized s and ﬂushed embryos.

ES Cell derivation ol gsuperovulationz Day 0: 1) Euthanized female rat with C02. 2) Swabbed ventral abdomen with 70% ethanol; using scissors, opened the ventral body wall to expose the viscera. 3) Dissected out the oviducts and uterine horns and placed them into a tissue e dish containing warm N2B27 media. Washed out as much blood as possible and transferred to a new dish with N2B27. 4) Using a 1 ml syringe and a blunt 27g , ﬂushed media through the uterine horns and oviducts to eject blastocysts into the media. ) ted the blastocysts with a mouth pipet and transfer to embryo culture dish containing KSOM + 2i (1 uMPD032590l, 3 uM CHIR99021). KSOM is a culture medium produced by Millipore. Catalog number is MR-lO6-D. 6) Cultured overnight at 37°; 7.5% C02.

ES Cell derivation protocol gfrozen embryos! Day 0: 1) Thawed frozen 8-cell embryos (commercially obtained) into M2 medium.

Cultured 10 minutes at room temperature. 2) Transferred to KSOM + 2i and culture overnight.

ES Cell derivation protocol gsame for both) Day 1: 1) Transferred cavitated embryos to 2i medium & culture overnight. 2) Continued culturing un-cavitated embryos in KSOM +2i Day 2: 2014/017452 1) Transferred all remaining embryos to 2i medium (whether or not they've cavitated). 2) Cultured overnight; continued culturing earlier embryos in 2i medium.

Day 3: 1) Transferred embryos for 30 — 60 s with Acid Tyrodes to remove the zona pellucida. 2) Washed embryos 3X in 2i medium to remove Acid Tyrodes. 3) ted each embryo into a separate well of a 96-well feeder plate (the well contains a monolayer of mitotically vated mouse embryonic fibroblasts (MEFs). 4) Cultured overnight in 2i medium.

Day4—5: 1) Monitored plated embryos for the presence of an wth (an amorphous undifferentiated mass of . Outgrowths are ready for transfer when they are approximately twice the size of the plated embryo. 2) Each day: remove spent media with a mircropipet and replace with fresh 2i media. 3) Transferred outgrowths to new feeder wells: a. Removed spent media and gently wash well with PBS. b. Removed PBS and add 30 ul 0.05% trypsin; incubate for 10 minutes. c. Stopped trypsin reaction by adding 30 ul 2i + 10% FBS. d. Gently iated the cells with a micropipettor and transferred entire contents of the well to a new well in a 24-well feeder plate.

This was Passage 1 (Pl). e. Cultured overnight in 2i medium.

Day 5 — 8: (timing depends on how fast each line expands) 1) Changed media each day (2i media) and monitored for the presence of colonies with an ESC morphology. 2) When colonies , continued culturing until colonies expand to ~ 50% conﬂuency. 3) Tryspinzed and passaged colonies as before; plated on feeders, 1 well per line, in a 6-well dish. This was Passage 2 (P2).

Ongoing: 1) Continued feeding and monitoring each line until approximately 50% 2) Trypsinized cells as usual. 3) stopped trypsin with 2i + 10% FBS; ed the cells by centriﬁJgation (5’, 1200 rpm in Beckman-Coulter tabletop centrifuge). 4) Aspirated the supernatant and gently resuspend the cells in 400 pl Freezing Medium (70% 2i, 20% PBS, 10% DMSO).

) Distributed the cells into 2 Vials and freeze at -80°. This was Passage 3 (P3). 6) For erm storage, transferred the Vials to liquid N2 storage.

The 2i media was prepared as follows in Table 7.

Reagent Vendor Concentration DMEM/F l2 basal media InVitrogen/Life lx Technologies Neurobasal media Invitrogen/Life lx Technologies Penicillin/streptomycin InVitrogen/Life Technologies L-Glutamine InVitrogen/Life logies aptoethanol InVitrogen/Life Technologies N2 supplement Invitrogen/Life lx Technologies B27 supplement InVitrogen/Life lx Technologies LIF Millipore 100 U/ml PD0325901 (MEK Stemgent 1 uM inhibitor) .

CHIR9902l (GSK Stemgent 3 uM inhibitor) .

Materials: Pregnant Mare’s Serum Gonadotropin (PMSG) Human Pregnancy Urine Chorionic Gonadotropin (HCG) Female Rats (5-12 weeks old) Male rats (12 wks. to 8 mos. old), one per cage Syringes/needles Animal room with lights on 6:00-18:00 Procedure: Day 1: 8:00-10:00 AM Inject females with 20 IU PMSG (0.4 ml), IP Discard unused PMSG.

Day 3: 8:00-10:00 AM (48 hours after PMSG injection) Inject females with 50 IU HCG (1 ml), IP Place one female per male in mating cage.

Discard unused HCG.

Day 4: 8:00-10:00 AM (24 hrs. after HCG injection) Check females for plugs.

Hormone ers PMSG: Sigma #G-4877 (1000 IU). Resuspend in PBS to a ﬁnal [ ] of 50 IU/ml.

Store at -20° in 1 ml ts.

HCG: Sigma #CG-5 (5000 IU). end in PBS to a ﬁnal [ ] of 50 IU/ml.

Store at -20° in 1 ml aliquots.

Example 4: yping of Rat Embryonic Stem Cell Lines The rat ES cell line generated herein were karyotyped, and the results are summarized in Tables 8-1 1.

Table 8 ACI.G1 Kar 0 in_ Results Number of cells Number of cells karyotyped 7 Number of cells analyzed 20 Number of 42, XY cells 18 Number of abnormal cells 2 40, XY, -5, -9 1 41, XY, -14 1 42, XY 18 Other notes: Two analyzed cells were missing different autosomes, which may be a sporadic occurrence due to technical ct. 90% of analyzed cells had a normal male 42, XY karyotype.

Figure 9 provides a photograph showing the analysis of the chromosome number of the ACI.Gl rat ES cell line.

Table 9 DA.2B Kar 0 in_ Results Number of cells Number of cells karyotyped 6 Number of cells analyzed 20 Number of 42, XY cells 20 Number of abnormal cells 0 42, XY 20 Other notes: All analyzed cells had a normal diploid 42, XY karyotype.

Figure 10 provides a photograph g the analysis of the chromosome number of the DA.2B rat ES cell line.

Table 10 DA.C2 Kar 0t in_ Results Number of cells Number of cells karyotyped 5 Number of cells analyzed 20 Number of 42, XY cells 20 Number of abnormal cells 0 42, XX Other notes: 100% of analyzed cells had normal female XX rat ype.

Figure 11 provides a photograph showing the analysis of the chromosome number of the DA.C2 rat ES cell line.

Table 11 Lines Lines Blastocyst Karyotype s plated estabdlishe Karyotypes BN x SD all lines were high % complex F1 8 (20%) polyploid G1: 90% 42 XY; others were 70-85% 16W) euploid -—-—2B: 100% 42 XY; 2C: 100% 42 ers were 95-100% euploid -—--_— Totals 92 34 (37%) Example 5: Electroporation of Vector into Rat Embryonic Stem Cell 1. Passaged rat ES cells 24-48 hrs prior to electroporation. 2. Changed media to RVG2i + ROCKi (10uM 2) 24 hr. prior to electroporation 3. Changed media 30’ prior to trypsinization.

. Aliquoted DNA to be electroporated. d DNA to warm at RT for >10 min.

. Heated DNA for 5’ @ 62°C. Place DNA on ice.

Trypsinized cells: a. Collected ﬂoating colonies. Washed plate to collect as many ﬂoaters as possible.

Pelleted colonies: 3’ @ 750 rpm.

Washed pellet 1X with 5-10m1 PBS and re-spin/pe11et ted supernatant; add 500% trypsin, 0.05% + 1% chicken serum. i. Did not p001 more than 1 10 cm plate of colonies per tube. If there are too many colonies packed into the bottom of the tube during trypsinization they will clump and most of the cells will be lost. 6. 4’ @ 37°. Pipeted colonies l times to minimize clumping. f. Repeated steps 1-2 X: 4’ @ 37°. g. Stopped trypsin with 500% RVG2i + 10% PBS. 8. Pelleted cells: 5’ @ 1200 rpm. 9. Resuspend cells in 10 m1 PBS. Count two 20% aliquots to determine total cell number.

. Pelleted cells (5’/1200rpm); calculate total cell number and total resuspension volume to achieve correct cell concentration (target #/75 ul EP buffer). 11. Resuspend in a minimal volume of EP buffer; measure total volume and adjust to target volume with EP . Electroporation buffer is sold by Millipore. The catalog # is ESD. See, Valenzuela et al. (2003) Nature Biotechnology 21 :652-659, which is herein incorporated by reference. 12. Add 751 cells to 507t DNA; transfer the 125K ce11s/DNA solution to one well of a BTX l cuvette. a. Filled the empty wells in the same column with 1251 EP . 13. Pulsed the cuvette once in the BTX electroporator: WO 30706 a. Settings: 400V; 9; 100 uF (settings may vary) 14. Placed cuvette on ice for 15’ to r.

. Removed cells into 5 ml RVG2i + 10uM ROCKi. 16. Added to 15 cm plate with 20 ml RVG2i + 10uM ROCKi. Plate has 2X neoR MEFs (or other MEFs depending on project). The neoR selectable marker is the neomycin phosphotransferase (neo) gene of Beck et al. (1982) Gene, 19:327-36 or in US Patent No, 148 or 6,596,541, each of which are herein incorporated by reference. 17. Incubated @ 37°. Begin selection 48hrs later.

ROCK inhibitor used was Y-27632.

Example 6: Selecting Targeted Genetic Modiﬁcation in a Rat Embryonic Stem 1. Passaged cells for 24-48 hrs prior to electroporation. 2. Changed media to RVG2i + ROCKi (10uM Y-27632) 24 hr. prior to electroporation 3. Changed media 30’ prior to nization. 9.09:“ Aliquoted DNA to be electroporated.

Allowed DNA warm at RT for >10 min.

Heated DNA for 5’ @ 62°C. Place DNA on ice.

Trypsinized cells: h. Collected g colonies. Washed plate to collect as many ﬂoaters as possible. i. Pelleted colonies: 3’ @ 750 rpm. j. Washed pellet 1X with 5-10ml PBS and re-spin/pellet k. Aspirated supernatant; add 500% trypsin, 0.05% + 1% chicken serum. i. Did not pool more than 1 10 cm plate of colonies per tube. If there are too many colonies packed into the bottom of the tube during trypsinization they will clump and most of the cells will be lost. 1. 4’ @ 37°. Pipeted colonies several times to minimize clumping m. Repeated 1-2 X: 4’ @ 37°. 11. Stopped trypsin with 500k RVG2i + 10% PBS. 8. Pelleted cells: 5’ @ 1200 rpm. 9. ended cells in 10 ml PBS. Count two 20% aliquots to determine total cell number.

. Pelleted cells (5’/1200rpm); calculate total cell number and total resuspension volume to achieve correct cell concentration t #/75 ul EP buffer). 11. Resuspend in a minimal volume of EP buffer; measured total volume and adjusted to target volume with EP buffer. 12. Added 75% cells to 50% DNA; transfer the 125k cells/DNA solution to one well of a BTX 48-well cuvette. a. Filled the empty wells in the same column with 1251 EP buffer. 13. Pulsed the cuvette once in the BTX electroporator: a. Settings: 400V; 400V; 9; 100 uF (settings may vary) 14. Placed cuvette on ice for 15’ to recover.

. Removed cells into 5 ml RVG2i + 10uM ROCKi. 16. Added to 15 cm plate with 20 ml RVG2i + 10uM ROCKi. Plate had 2X neoR MEFs (or other MEFs depending on project). 17. Incubated @ 37°. Began ion 48hrs later. 18. G418 ion protocol was as follows: a. Day 2 (211d day after EP): incubated cells in 2i media + G418, 75 ug/ml. b. Day 3: incubated cells in 2i media without G418 c. Day 4: incubated cells in 2i media + G418, 75 ug/ml. d. Day 5: incubated cells in 2i media t G418 e. Day 6: incubated cells in 2i media + G418, 75 ug/ml. f. Day 7: incubated cells in 2i media without G418 g. Day 8: incubated cells in 2i media + G418, 75 ug/ml. h. Day 9: incubated cells in 2i media without G418 i. Day 10: incubated cells in 2i media + G418, 75 ug/ml. j. Day 11: incubated cells in 2i media without G418 k. Day 12: picked colonies to expand for screening. Each colony was dissociated in 0.05% trypsin + 1% chicken serum for 10 minutes and then plated into 1 well of a 96-well feeder plate. 19. Expanded colonies for 3 days in 2i media. . ed clones 1:1 to new 96-well feeder plates.

WO 30706 21. Expanded clones for 3 days in 2i media. 22. For each clone, dissociated colonies in trypsin. Froze 2/3 of each clone and store at -80°; plated the remaining 1/3 onto laminin plates (96-well plates coated with 10 ug/ml laminin). 23. When the laminin plates were conﬂuent, passed off to the screening lab for genotyping of the clones.

Example 7. Molecular Signature of the Rat Embryonic Stem Cells The genes listed in Table 13 were expressed at levels 20-fold higher in rat ES cells than the corresponding genes in mouse ES cells. The genes listed in Table 12 were expressed at d lower in rat ES cells than the corresponding genes in mouse ES cells.

The microarray data in Tables 12 and 13 was generated as follows. Rat ES cells (ACI.G2 and DA.2B) and mouse ES cells (FlH4) were cultured in 2i media for 3 passages until conﬂuent. FlH4 cells were cultured on gelatin-coated plates in the absence of feeders. FlH4 mouse ES cells were derived from 129S6/SvaTac and C57BL/6NTac heterozygous embryos (see, e.g., US Pat. No. 7,294,754 and Poueymirou, W.T., Auerbach, W., Frendewey, D., Hickey, J.F., Escaravage, J.M., Esau, L., Dore, A.T., Stevens, 8., Adams, N.C., Dominguez, M.G., Gale, N.W., Yancopoulos, G.D., DeChiara, T.M., Valenzuela,D.M. (2007), incorporated by nce herein in its entirety).

The following protocol was used for sample prep: Materials included TRIzol Reagentp; RNA Lysis Buffer (Zymo Kit); and 1.5 mL orf tubes.

The 1.5mL Eppendorf tubes were labeled with the Sample ID. Cells grown on a plate were rinsed in 37C PBS. PBS was removed and 300 ul of Trizol was added. A scraper was used to break the cells in Trizol. The lysed cells were collected in Trizol in a 1.5mL orf tube. For cells grown on suspension, the cells were rinsed in 37C PBS.

The cells were collected in a 1.5mL tube, the cells were spun down, PBS was d and 300 ul of Trizol was added. The cells were pipeted up and down to break the cells.

Samples were sorted for FACS with 101 to 105 cells, the volume was trated to less than 100uL. 4 volumes ofRNA Lysis buffer was added and mix by pipetting. For , 320uL RNA Lysis buffer was added to 80uL sample. Samples were stored at — °C.

RNA-Seq was used to measure the expression level of mouse and rat genes.

Sequencing reads were mapped to mouse and rat reference genome by Tophat, and RPKM (fragments per kilobase of exon per million fragments mapped) were calculated for mouse and rat genes. gy genes based on gene symbol were selected, and then used t-test to e gene's expression level between mouse and rat. miR-632 is in the top 10 highest expressed in rat ESCs but they were not sed in mouse ES cells. There is ore no comparative data for these genes.

Based on the levels of expression compared to other genes and their known function in the embryonic pment, the expression of miR-632 were used as a marker for rat ES cells.

Table 12. The genes listed were expressed at levels 20-fold lower in rat ES cells than the corresponding genes in mouse ES cells.

ID Notes Symbol Type<s> Dmg<s> ATP-binding cassette, sub- family B Abcb (MDR/TAP), Plasma lb Abcb lb member 1B Membrane transporter smooth , Acta2 ACTA2 aorta Cytoplasm other smooth muscle, Actg2 ACTG2 enteric Cytoplasm other 1 AEBPl 1 Nucleus peptidase W ANG”_- l2 angiopoietin-like 2 Space other ankyrin repeat Ankrd ANKRD domain 1 (cardiac transcriptio l 1 muscle) Cytoplasm n regulator Anxa Plasma 1 ANXAl annexin A1 Membrane other hydrocortisone 6 ANXA6_-annexin A6 Membrane other ANXA8 Anxa (includes Plasma 8 others) annexin A8-like 2 Membrane other Rho guanine nucleotide Arhge ARHGE exchange factor £25 F25 (GEF) 25 Cytoplasm other Axl -_-AXL tyrosine kinase Membrane kinase cabozantinib Baspl transcriptio signal protein 1 Bgn BGN _-biglycan Space other bone marrow stromal cell antigen Plasma Bst2 BST2 2 Membrane other transcriptio Btf3 -—BTF3 factor 3 Nucleus n regulator BTG family, transcriptio Btg2 BTGZ member 2 Nucleus n regulator Capsl CAPSL hosine-like Other other -caveolin l, transmemb caveolae protein, Plasma rane CaVl CAVl 22kDa ne receptor Ccdc8 -_CCDC80 containing 80 A5:0~('D5:m other Ccnd A5:O~(I)5:m other Cd24 CD248 endosialin VIembrane other CD44 molecule (Indian blood Plasma Cd44 CD44 group) VIembrane enzyme G-protein coupled Cd97 --CD97 CD97 molecule VIembrane receptor CDC42 effector Cdc4 CDC42E protein (Rho 2ep5 P5 GTPase binding) 5 asm other cadherin 11, type 2, thl OB-cadherin Plasma CDHl l (osteoblast) Membrane other Cdkn transcriptio A kinase inhibitor 2A Nucleus n regulator Cdo l -mCDOl diox enase t .e l C o .lasm enz me CAP-GLY domain containing linker - Clip3 -CLIP3 protein 3 Cytoplasm other lipofuscinosis, Cln5 CLN5 neuronal 5 Cytoplasm other calponin 1, basic, Cnnl CNNl smooth muscle Cytoplasm other collagenase Colla collagen, type I, Extracellular clostridium COLlAl alpha 1 Space other histolyticum collagenase Colla collagen, type I, Extracellular idium COLlAZ alpha 2 Space other histolyticum collagenase Col3a collagen, type III, Extracellular clostridium COL3A1 al .ha 1 S 0 ace other histol icum collagenase ColSa en, type V, Extracellular clostridium COLSAZ alpha 2 Space other histolyticum Col6a collagen, type VI, Extracellular collagenase COL6A2 alpha 2 Space other clostridium histolyticum CRYAB c stallin, alnha B other colony stimulating factor 1 Extracellular Csfl CSFl (macrophage) Space cytokine cystathionase (cystathionine Cth O mT gamma-lyase) Cytoplasm enzyme collagen triple Cthrc helix repeat Extracellular CTHRCl containing 1 Space other Ctsc CTSC Cytoplasm peptidase cysteine-rich, angiogenic inducer, Extracellular CYR6 l 6 1 Space other DEAD (Asp-Glu- Ala-Asp) box DDX58 polypeptide 58 asm enzyme dickkopfWNT signaling pathway DKK3 inhibitor 3 S ace c okine DMCl —-recombinasel Nucleus enzyme DPYSL3 —-e-like3 Cytoplasm enzyme DSE _-epimerase Cytoplasm enzyme dual speciﬁcity - phosphatas DUSPl atase 1 Nucleus e dual speciﬁcity phosphatase 27 phosphatas DUSP27 (putative) Other e dual speciﬁcity atas DUSP9 phosphatase 9 Nucleus e endothelin converting enzyme Plasma Ece2 ECEZ 2 Membrane peptidase Ecml ECMl protein 1 Space orter transcriptio EGRl _-resonsel Nucleus n reulator epithelial membrane protein Plasma Emp l EMF 1 l Membrane other epithelial membrane protein Plasma Emp3 EMP3 3 Membrane other Ephx epoxide hydrolase EPHXZ 2, cytoplasmic Cytoplasm enzyme coagulation factor transmemb activated 111 (thromboplastin, Plasma rane inant F3 ’11 U.) tissue ) Membrane receptor human factor VII Finkel-Biskis- Reilly murine sarcoma virus (FBR-MuSV) tously Fau FAU expressed Cytoplasm other Extracellular an1 FBN1 ﬁbrillin 1 Space other Fbxol transcriptio FBXO l 5 F-box protein 15 Other n regulator four and a half transcriptio FhlZ FHLZ LIM domains 2 Nucleus n tor Flnc FLNC ﬁlamin C, gamma Cytoplasm other FBJ murine osteosarcoma Viral transcriptio Fos FOS oncogene homolog Nucleus n regulator Fundc FUNDC FUN 1 4 domain 2 containing 2 Cytoplasm other gap junction protein, beta 3, Plasma Gjb3 GJB3 3 1kDa ne transporter Gpa3 Plasma GPA3 3 (transmembrane) Membrane other GC-rich promoter prp GPBP1L binding protein 1- 111 1 like 1 Other other Plasma Gpc3 GPC3 Membrane other growth factor receptor-bound Grb 1 0 C) ._. O protein 10 Cytoplasm other Gstm GSTMS transferase mu 5 Cytoplasm enzyme huntingtin- associated protein Hap 1 HAP 1 1 Cytoplasm other HISTZH Hist1 (includes histone cluster 2, h2bc others) H2be Nucleus other nga high mobility HMGAZ ,urou AT-hook 2 Nucleus Cl’lZ me high mobility ngn group nucleosomal ngn3 binding domain 3 s other Horm HORMA HORMA domain ad1 containing 1 Nucleus other hydroxysteroid Hsdl HSD17B ( l 7 -beta) 7b 1 4 14 dehydrogenase 14 asm enzyme Hspb HSPB1 protein 1 Cytoplasm other Hspb HSPB8 protein 8 asm kinase Extracellular Htra1 HTRAl peptidase 1 Space peptidase Iﬁ204 des eron activated transcriptio Iﬁ204 others) gene 204 Nucleus n regulator interferon-induced Iﬁ44 IFI44 0 rotein 44 C o .lasm other interferon-induced protein with tetratricopeptide Iﬁtl IFIT1B repeats 1B Cytoplasm other interferon induced If1tm3 IiEﬁ-ﬁﬁiillll transmembraneIFITMZ protein 2 Cytoplasm other insulin-like growth factor 2 Extracellular growth Igf2 IGFZ (somatomedin A) Space factor insulin-like growth Igfbp factor binding Extracellular IGFBP7 protein 7 Space transporter transmemb interleukin 1 Plasma rane ll 1 rl l ILlRLl receptor-like l ne rece tor Inhba HBA ibin,betaA S 0 ace factor Inhbb HBB —-—inhibin,betaB Space factor Irf7 regulatory factor 7 Nucleus n regulator Isng 3 _-—likemodifier Space other integrin, alpha 5 (fibronectin transmemb receptor, alpha Plasma rane Itga5 ITGAS polypeptide) VIembrane receptor _-—junproto-oncogene \Iucleus n regulator Junb U3 _—-oncogene us n regulator lectin, galactoside- transmemb Lgals LGALS3 binding, soluble, 3 rane 3bp binding n receptor Lgals lectin, galactoside- Extracellular LGALS9 , soluble, 9 Sace other Lmna LMNA Extracellular Lox LOX l s loxidase Sace enz me Lole LOXLZ yloxidase-like2 Space enzyme Loxl3 LOXL3 _-—lysyloxidase-like 3 Space enzyme low density lipoprotein transmemb receptor-related Plasma rane Lrpl protein 1 Membrane receptor Mage MAGEB b1 6 l 6 family B, 16 Other other Mcam MCAM _-—adhesionmolecule Membrane other MGP _-—matrixGlaprotein Space other matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV Extracellular MMP2 collagenase) Space ase marimastat matrix-remodelling E><RA8 associated 8 Other other Myl9 MYL9 9, regulatory Cytoplasm other myosin light chain, phosphorylatable, Mylpf MYLPF fast skeletal muscle Cytoplasm other NGFI-A binding protein 2 (EGRl transcriptio Nab2 Z binding protein 2) Nucleus n regulator NADH ogenase (ubiquinone) 1 beta Ndufb NDUFB subcomplex, 4, 4; l SkDa C o .lasm trans 0 orter nucleophosmin (nucleolar phosphoprotein transcriptio Npml NPMl B23, numatrin) Nucleus n regulator ligand- nuclear receptor ent subfamily 0, group nuclear Nr0bl NROBl B, member 1 Nucleus or ligand- nuclear or dependent subfamily 4, group nuclear Nr4al NR4Al A, member 1 Nucleus rece tor Plasma Nrp2 Z Membrane kinase 2'-5'-oligoadenylate synthetase l, Oasla OASl Da Cytoplasm enzyme 2'-5' denylate Oale Oale synthetase-like 2 Other enzyme prolyl 4- hydroxylase, alpha P4ha2 P4HA2 polypeptide 11 Cytoplasm enzyme poly ibose) polymerase family, Parp3 PARP3 member 3 Nucleus enzyme procollagen C- Pcolc endopeptidase Extracellular PCOLCE enhancer Space other phosphate Pcytl cytidylyltransferase PCYT lB l, choline, beta Cytoplasm enzyme Extracellular growth Pdfc PDGFC rowth factor C S u ace factor pleckstrin homology-like Phlda domain, family A, PHLDAl member 1 Cytoplasm other pleckstrin homology-like Phlda domain, family A, PHLDAZ member 2 0‘5plasm other phospholipase A2, Pla2g PLAZGl group IB Extracellular lb B (pancreas) Space enzyme niflumic acid Pla2g PLA2G4 phospholipase A2, A group IVA Cytoplasm enzyme quinacrine WO 30706 (cytosolic, dependent) Porcn -_-PORCN (Drosophila) Cytoplasrn periostin, osteoblast speciﬁc Extracellular Postn POSTN factor Space Prrxl PRRXl_—-homeoboxl Nucleus n regulator 3 PRSSZ3 protease, serine, 23 Space ase some (prosorne, rnacropain) Psrnb subunit, beta type, 8 PSMB8 8 Cytoplasrn peptidase acetaminophen/pe ntazocine, acetaminophen/cl emastine/pseudoe phedrine, aspirin/butalbital/ caffeine acetaminophen/ca ffeine/dihydrocod eine, aspirin/hydrocodo aspirin/oxycodone acetaminophen/as pirin/caffeine, aspirin/praVastatin acetaminophen/de phenirarnin e/pseudoephedrin aspirin/meprobarn ate, n/caffeine/pr opoxyphene, aspirin/butalbital/ caffeine/codeine, aspirin/caffeine/di hydrocodeine, chlorphenirarnine/ ibuprofen/pseudoe phedrine, licofelone, menatetrenone, icosapent, suprofen, prostaglandin— lornoxicarn, endoperoxide tiaprofenic acid, synthase 2 lurniracoxib, aglandin G/H tenoxicarn, synthase and naproxen/surnatri Ptgs2 PTGSZ cyclooxygenase) Cytoplasrn ptan, ibuprofen/phenyle phrine, acetaminophen/as pirin/codeine, naproxen/esomepr azole, famotidine/ibupro fen, ibuprofen/phenyle phrine/chlorphenir amine enac/misopr 0st01, acetaminophen/bu talbital/caffeine, hydroc0d0ne/ibup r0fen, acetaminophen/by droc0d0ne, acetaminophen/Ha madol, acetaminophen/c0 deine, acetaminophen/0X ycodone, acetaminophen/pr opoxyphene, niﬂumic acid, nitroaspirin, ofen, diclofenac, etoricoxib, naproxen, meclofenamic acid, pomalidomide, meloxicam, celecoxib, dipyrone, nimesulide, acetaminophen, mefenamic acid, sal, ibuprofen, GW4063 8 1X, phenylbutazone , indomethacin, sulfasalazine piroXicam, valdecoxib, aspirin, carprofen, zomepirac, r0fecoxib, n/methocarb amol, aspirin/caffeine/or phenadrine , aspirin/carisoprod aspirin/carisoprod III%EIIIE[HI— 01/codeine, acetaminophen/bu talbital, balsalazide, aspirin/dipyridarn ole, inophen/bu talbital/caffeine/c odeine, racemic ﬂurbiprofen, phenacetin, sulindac, nabumetone, tolrnetin, ketorolac, oxaprozin, mesalarnine, salsalate, fenoprofen, salicylic acid, inophen/ch lorphenirarnine/hy drocodone/phenyl ephrine/caffeine, bromfenac Extracellular growth Ptn PTN pleiotrophin Space factor polymerase I and transcript release transcriptio Ptrf W factor Nucleus n regulator etretinate, adapalene, isotretinoin, ligand- tazarotene, dependent acitretin, tretinoin, retinoic acid r alitretinoin, Rarg receptor, gamma Nucleus rece tor fenretinide regulator of G- protein signaling Rgsl6 7‘ 1 6 Cytoplasrn other 45S pre-ribosornal Rn45s E45s RNA Other other RpllO aL 1 0A L10a Other other Rpl3l aL31 L31 Other other Rpl37 a L3 7a Cytoplasrn other Rple z rough Cytoplasrn other translation Rpsl4 aS14 S14 Cytoplasrn regulator R. 820 Rs20 S20 C o n lasrn other Rps26 aS26 S26 Cytoplasrn other ribosomal protein translation Rps9 E S9 asrn regulator WO 30706 SlOOa 4 S100A4_-binding protein A4 Cytoplasm other SlOOa 6 S100A6_-binding protein A6 Cytoplasm transporter schwannomin Sdnp interacting protein 1 SCHIPl l Cytoplasm other Plasma Sdc2 SDCZ syndecan 2 Membrane other serpin peptidase tor, clade E (nexin, nogen Serpi SERPIN activator inhibitor Extracellular nel ,_. type 1), member 1 Space co in alfa serpin peptidase inhibitor, clade E (nexin, nogen Serpi SERPIN activator inhibitor Extracellular ne2 mN type 1), member 2 Space other serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, t epithelium Serpi SERPIN derived factor), Extracellular H member 1 Space other Sh3gl SH3GL2 GRBZ-like 2 Membrane enzyme solute carrier family 19 (thiamine Slcl9 SLC19A transporter), Plasma a2 member 2 Membrane transporter solute carrier family 25 (mitochondrial carrier; adenine nucleotide Slc25 SLCZSA translocator), a5 member 5 Cytoplasm trans orter clodronic acid solute carrier family 29 (equilibrative nucleoside SLC29A transporter), Plasma member 1 Membrane transporter solute carrier SLC35F family 3 5, member N F2 Other other small r ribonucleoprotein SNRPN polypeptide N Nucleus other SNX22 transporter secreted protein, acidic, cysteine- Extracellular Sparc SPARC rich (osteonectin) Space other Sppl SPPl Extracellular cytokine phosphoprotein 1 Space sulfotransferase Su1t4a SULT4A family 4A, member 1 1 Cytoplasm enzyme Tagln TAGLN transgelin Cytoplasm other ription elongation factor A transcriptio Tcea3 TCEA3 (SH), 3 n regulator transforming growth factor, beta Extracellular growth Tgfb3 TGFB3 3 Space factor Thbsl THBSl _-thrombospondin1 Space other _Extracellular Thbs2 THBSZ thrombospondin 2 Space other transmembrane 4 L Tm4s six family member Plasma TM4SF1 1 Membrane other transmembrane - Tmbi TMBIM BAX inhibitor 1 motif containing 1 Cytoplasm other m176 TMEMl b 76B ein176B Other other Tnc TNC _-tenascinC Space other Tpd5 TPDSZL 211 1 _-like1 Cytoplasm other Tme TPMZ _-(beta) Cytoplasm other Usp 1 tin specific 8 USP18 ase 18 Cytoplasm peptidase Vim VIM Cytoplasm other WAP four- Wfdc disu1f1de core Extracellular 2 WFDCZ domain 2 Space other WNTl inducible Wisp signaling pathway Extracellular growth 2 WISPZ protein 2 Space factor Y box binding transcriptio bel YBX 1 protein 1 n regulator Table 13. The genes listed were expressed at levels 20-fold higher in rat ES cells than the corresponding genes in mouse ES cells.

Entrez Gene Name on Type(S) Dmg(S) adherens on associated protein 1 other adenosylmethionine decarboxylase 1 Cytoplasm enzyme ankyrin repeat domain 2 (stretch transcription responsive muscle) regulator Cdc42 guanine nucleotide exchange factor (GEF) 9 Cytoplasm other ATP synthase, H+ transporting, mitochondrial F0 Atp5h complex, subunit d Cytoplasm enzyme Btg3 BTG3 member 3 Nucleus other olamide, hydrochlorothiazi de, olamide, trichloromethiazid e, chlorothiazide, chlorthalidone, benzthiazide, carbonic anhydrase Extracellul sulfacetamide, Car6 VI ar Space enzyme topiramate calcium/calmodulin- dependent protein Camk4 CAMK4 kinase IV Nucleus kinase Capnlz___— chaperonin containing TCPl, Cct6b CCT6B subunit 6B (zeta 2) asm transporter Cdx2 CDXZ homeobox 2 Nucleus regulator Cldn5 ___-CLDNS claudin 5 Membrane other C—type lectin domain family 3, Clec3a CLEC3A member A Other other - de intracellular_- Clic6 CLIC6 channel 6 Membrane ion channel dehydrogenase/redu Dhrsx I DHRSX X-linked r enzyme Dpyle DPYSLZ -like 2 Cytoplasm enzyme dual speciﬁcity phosphatase 26 Dusp26 DUSP26 (putative) Cytoplasm enzyme Eci3 Eci3 delta isomerase 3 Other enzyme eukaryotic elongation factor-2 Eeka EEFZK kinase Cytoplasm kinase Efnal ___-EFNAl ephrin-Al Membrane other Epha4 ___-EPHA4 EPH receptor A4 Membrane kinase ﬁbronectin type III and ankyrin repeat transcription Fankl FANKl domains 1 Nucleus regulator Fhit FHIT triad asm enzyme Filipl FILIPl interacting protein 1 Cytoplasm other Fmod ___-FMOD ﬁbromodulin ar Space other Foxel FOXEl (thyroid Nucleus regulator —-2) Fry FRY (Drosophila) ar Space other gap junction protein, Gjbs GJBS beta 5, 3 l . lkDa Membrane transporter glutathione - peroxidase 2 pr2 ointestinal) Cytoplasm enzyme GRXCR glutaredoxin, - ercr2 2 cysteine rich 2 Other other HECT, C2 and WW domain containing E3 ubiquitin protein Extracellul Hecw2 liase 2 ar Snace Cl’lZ The enhancer-of— split related with transcription Hey2 YRPW motif 2 Nucleus regulator inducible T-cell co- Icos Icos stimulator Membrane interferon induced transmembrane Plasma transmembra Ifitml IFITMl protein 1 Membrane ne rece tor Illf‘8 Interleuk in 3 6 beta 112 8ra Interleuk in 2 8 receptor, alpha n-like growth IGFBPL factor binding I_nﬂ)ll 1 urotein—like 1 Other other interaction protein for cytohesin Ipcefl IPCEFl exchange factors 1 Cytoplasm enzyme Lctl Lctl lactase-like Cytoplasm other lactate thd LDHD dehydrogenase D Cytoplasm enzyme lymphoid enhancer- transcription Lefl LEFl binding factor 1 Nucleus regulator left-right determination factor Extracellul Leftyl LEFTYl 1 ar Space growth factor leukemia tory transmembra Lifr LIFR factor receptor alpha Membrane ne receptor G-protein lysophosphatidic Plasma coupled Lpar2 acid receptor 2 Membrane rece tor myelin oligodendrocyte ellul Mog glycoprotein ar Space other MORN repeat Morn5 MORNS nin; 5 Other other nuclear cap binding protein subunit 2, Pigz 20kDa Nucleus other 2014/017452 neuronal pentraXin Plasma transmembra Nptxr Ntm —--NTM neurotrimin Membrane other nuclear transport -- Nutf2 NUTFZ factor 2 Nucleus transporter Ocln OCLN occludin --Membrane enzyme oxidized low density lipoprotein (lectin- Plasma transmembra Olr l like) receptor 1 Membrane ne receptor poly(A) binding protein, cytoplasmic translation Pabpc4 PABPC4 4 (inducible form) Cytoplasm regulator dyphylline, nitroglycerin, aminophylline, dipyridamole, tolbutamide, tadalafil, phosphodiesterase theophylline, Pdel l a PDEl 1A 1 1A Cytoplasm enzyme pentoxifylline den PDYN prodynorphin ar Space transporter period circadian Per3 PER3 clock 3 Nucleus other Pllp —“as“PLLP plasmolipin ne transporter protein atase 1, regulatory Ppplrl4 PPP 1 R1 4 (inhibitor) subunit O C 14C Cytoplasm other preferentially expressed antigen in Pramel6 Pramel6 melanoma like 6 Other other protein ne phosphatase, non- receptor type 18 Ptpnl 8 PTPNl 8 -derived) Nucleus phosphatase pyrroline-S - carboxylate Pycr l reductase l Cytoplasm enzyme RAB26, member RAS oncogene Plasma Rab26 family ne enzyme receptor (G protein- - coupled) activity Plasma Ramp2 RAMPZ modifying protein 2 Membrane transporter pramlintide RNA binding motif - Rbm24 RBM24 protein 24 Other other Rh-associated E RHAG glycoprotein Membrane ase ribosomal n - rringtonin Rpl3 RPL3 L3 Cytoplasm other C sal-like 3 - Sall3 SALL3 (Drosophila) Nucleus other transcription Satb l SATBl SATB ox l -Nucleus regulator ch2 SCGZ secretogranin II cytokine solute carrier family (oligopeptide SLC15A orter), Plasma Slc15a1 1 member 1 Membrane transporter solute carrier family (neuronal/epithelial high ty glutamate transporter, system Plasma Slc1a1 SLC1A1 Xag), member 1 Membrane transporter riluzole solute carrier family (sodium/potassium/c alcium exchanger), Slc24a5 member 5 Other other solute carrier family 37 (glucose phosphate SLC37A transporter), Slc37a2 2 member 2 Other transporter syntrophin, beta 1 (dystrophin- associated protein A1, 59kDa, basic Plasma 40424 comonent 1 Membrane other ST6 (alpha-N- acetyl-neuraminyl- 2,3-beta—galactosyl- 1,3)-N- acetylgalactosamini St6galn de alpha-2,6- ac3 transferase 3 asm enzyme Tex12 TEX12 testis expressed 12 s other Extracellul Tex15 TEX15 testis expressed 15 ar Space other transcription factor AP-Z alpha (activating er binding protein 2 transcription Tfap2a alpha) Nucleus regulator transmembrane Plasma Tmc1 TMC1 channel-like 1 Membrane other Tmem1 TMEM1 transmembrane 30 protein 130 Other other Tmem3 TMEM3 transmembrane 0b 0B protein 30B Other other translocase of outer mitochondrial Tomm2 membrane 20 homolog (yeast) Cytoplasm transporter TOX high mobility group box family Tox3 member 3 Other other tetratricopeptide th25 TTCZS repeat domain 25 Cytoplasm other thymidine Extracellul Tynm TYMP phosphorylase ar Space growth factor -—__— -_--_vesicle-associated -_--_Wfdcl2 core domain 12 ar Space other -—--WfdclSacore domain 15A Other other dc6a core domain 6A Other other Table 14. A subset of genes from Table 13 which are expressed at levels 20-fold higher in rat ES cells than the corresponding genes in mouse ES cells.

ID Entrez Gene Name Ajap1 Adheres Iunctions Associate Protein CldnS Claudin 5 Arhgef9 Cdc42 guanine tide exchange facter 9 Calcium/calmodulin- dependent protein kinase IV Efna1 -Al E oha4 EPH rece otor A4 Gjb5 gap junction protein beta Igfbpll Insulin-like growth factor bindin_ orotein-like 1 1128ra eukin 28 receptor, aloha Lefty1 left-right determination factor 1 Lifr Leukemia inhibitory factor rece otor aloha Lpar2 Lysophosphatidic acid receotor 2 rece otor Protein tyrosine phosphatase non- receotor oe 18 Fibronectin type III and ankyrin repeat domains Forkhead box E1 (thyroid transcription Hairy/enhancer-of—split related with YRPW motif id enhancer- bindin; factor 1 2014/017452 Sa113 Sal-like 3 hila SATB homeobox 1 An additional molecular signature employing the pluripotency markers/genes for the rat ES cells has also been developed. Table 15 provides a gene list and their sion ranks from the RNA proﬁling data. mRNA was isolated from rat ES cells and the expression level of various pluripotency markers were compared relative to each other. The "pluripotency genes" which are listed are genes that other groups have used (mostly in mouse, but also in rat) as markers of ES cells. Mesoderm endoderm and neural are similarly deﬁned. By "rank" is refers to the expression in our experiment: the higher the rank (1 is highest) the higher the expression. For example, Oct4's rank of 13 means that, of all the genes assayed, it was expressed higher than all but 12 genes.

Background in this experiment was any sion value below 30; 6107 genes had expression values of 30 or higher.

Table 15. Rat ES cell molecular ure employing the pluripotency, mesodermal, endodermal, neural and trohectoderm markers/genes.

Plurigotenc Plurigotenc Endoderma Endoderma yRank I lRank c—Myc 11195 Nestin 7761 Dnmt3L 11418 Pax6 13570 DppaZ Not tested 4571 30x2 681 DppaS Not tested Ecatl 9714 Eras Fbxols 6.13 Lefl LIF receptor Lin28 Nanog — ---- 55w ----_---- Stet. Notttsttt ---_---- Notttsted ---_---- All publications and patent applications mentioned in the speciﬁcation are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was speciﬁcally and dually indicated to be incorporated by reference. Unless otherwise apparent from the context of any embodiment, aspect, step or feature of the invention can be used in combination with any other. Reference to a range es any integers within the range, any ge within the range. Reference to multiple ranges includes composites of such ranges.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method for making a targeted c modification in rat embryonic stem (ES) cells, comprising: (a) culturing a population of rat ES cells under conditions comprising a layer of feeder cells that are not modified to express leukemia inhibitory factor (LIF) and a medium comprising about 50 U/mL to about 150 U/mL LIF and a combination of inhibitors ting of a MEK inhibitor and a GSK3 inhibitor; and (b) modifying the rat ES cells to comprise the targeted genetic modification, wherein the rat ES cells are capable of transmitting the targeted genetic modification through the germline.

2. The method of claim 1, further comprising generating the population of rat ES cells in step (a), comprising: (i) culturing in vitro a first layer of feeder cells that are not modified to s leukemia inhibitory factor (LIF) and a morula or a blastocyst stage rat , wherein the zona pellucida of the morula or blastocyst stage rat embryo has been removed, and wherein the culture conditions maintain pluripotency of a rat ES cell and comprise a medium comprising about 50 U/mL to about 150 U/mL LIF and a combination of inhibitors consisting of a MEK inhibitor and a GSK3 inhibitor; (ii) transferring an outgrowth of an amorphous undifferentiated mass of rat ES cells obtained from step (i) to an in vitro culture well comprising a second layer of the feeder cells that are not modified to s LIF; and (iii) ing the outgrowth under conditions comprising the medium comprising about 50 U/mL to about 150 U/mL LIF and a ation of inhibitors consisting of a MEK inhibitor and a GSK3 inhibitor.

3. The method of claim 1 or 2, wherein the rat ES cells: (ii) lack expression of c-Myc; and/or (iii) have a normal karyotype; and/or (iv) form spherical, free-floating es in culture.

4. The method of any one of claims 1-3, further comprising: (c) introducing at least one of the modified rat ES cells from step (b) into a rat host embryo to produce an F0 embryo; (d) implanting the F0 embryo into a surrogate ; (e) gestating the F0 embryo in the surrogate mother to term; and (f) identifying an F0 rat having the targeted genetic modification.

5. The method of claim 4, wherein the modified rat ES cell introduced into the rat host embryo is from the same rat strain as the rat host embryo.

6. The method of claim 4, wherein the modified rat ES cell introduced into the rat host embryo is from a different rat strain than the rat host embryo.

7. A method of making a genetically modified rat, comprising: (a) providing a rat ES cell line comprising a population of rat ES cells obtained by culturing isolated rat ES cells on a layer of feeder cells that are not modified to express ia inhibitory factor (LIF) with a medium comprising about 50 U/mL to about 150 U/mL LIF and a combination of inhibitors consisting of MEK inhibitor PD0325901 and GSK3 inhibitor CHIR99021, and wherein the population of rat ES cells: lacks expression of c-Myc; forms cal, free-floating es in e; is diploid; and is germline competent; (b) obtaining a rat ES cell clone comprising a targeted cation, wherein the obtaining consists of: (i) modifying the population of rat ES cells to comprise the targeted genetic modification; and (ii) identifying in a single g step a rat ES cell clone comprising the targeted genetic modification; (c) introducing the rat ES cell clone into a rat host embryo, wherein the rat ES cell clone is from a different rat strain than the rat host embryo; and (d) gestating the rat host embryo comprising the rat ES cell clone in a surrogate , wherein the surrogate mother produces F0 progeny comprising the targeted c modification, n the targeted genetic modification is transmitted through the germline.

8. The method of claim 7, wherein the providing step (a) ses: (i) culturing in vitro a first layer of the feeder cells that are not modified to express leukemia inhibitory factor (LIF) and a morula or a blastocyst stage rat embryo, wherein the zona pellucida of the morula or blastocyst-stage rat embryo has been removed, and wherein the culture conditions maintain pluripotency of a rat ES cell and comprise a medium comprising about 50 U/mL to about 150 U/mL LIF and a combination of inhibitors consisting of MEK inhibitor 901 and GSK3 tor CHIR99021; and (ii) transferring an outgrowth of an amorphous undifferentiated mass of rat ES cells to an in vitro culture well comprising a second layer of the feeder cells that are not modified to express LIF; and (iii) culturing the outgrowth under conditions comprising the medium comprising about 50 U/mL to about 150 U/mL LIF and a combination of inhibitors consisting of MEK inhibitor PD0325901 and GSK3 inhibitor CHIR99021, thereby maintaining pluripotency of the rat ES cells.

9. The method of any one of claims 4-8, further comprising breeding a male F0 rat comprising the targeted genetic cation with a wild type female rat to e an F1 progeny that is heterozygous for the targeted genetic modification;, and optionally breeding a male rat of the F1 progeny with a female rat of the F1 progeny to obtain an F2 progeny that is homozygous for the targeted genetic cation.

10. The method of claim 9, wherein at least 3% of the F0 rats having the targeted genetic cation transmit the targeted genetic modification to the F1 progeny, optionally n at least 10% of the F0 rats having the ed genetic modification transmit the targeted genetic modification to the F1 progeny, and optionally n at least 60% of the F0 rats having the targeted genetic modification transmit the ed genetic modification to the F1 progeny.

11. The method of any one of claims 1-10, wherein: (I) the rat ES cells are derived from an ACI rat or derived from a Dark Agouti (DA) rat; and/or (II) the rat ES cells are d from a morula stage or a blastocyst stage embryo from a superovulated rat.

12. The method of any one of claims 1-11, wherein the targeted genetic modification comprises an insertion, a deletion, a knockout, a knockin, a point mutation, or a combination thereof.

13. The method of claim 12, wherein the targeted genetic modification comprises an insertion of a logous polynucleotide into the genome of the rat ES cells.

14. The method of any one of claims 1-13, wherein the ing step comprises modifying the rat ES cells to comprise two or more targeted modifications, wherein the rat ES cells can transmit the two or more targeted genetic modifications through the germline.

15. The method of any one of claims 1-14, wherein the rat ES cells are male (XY) rat ES cells or female (XX) rat ES cells.

16. The method of any one of claims 1-15, wherein the rat ES cells have one or more of the following characteristics: (a) the rat ES cells express at least one pluripotency marker selected from Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, and Utf1; (b) the rat ES cells do not express one or more pluripotency markers selected from c-Myc, Ecat1, and Rexo1; (c) the rat ES cells do not express one or more mesodermal s selected from Brachyury and Bmpr2; (d) the rat ES cells do not express one or more endodermal markers selected from Gata6, Sox17, and Sox7; (e) the rat ES cells do not express one or more neural markers selected from Nestin and Pax6; (f) the rat ES cells express one or more pluripotency markers selected from Oct-4, Sox2, and alkaline phosphatase; and (g) the rat ES cells express of one or more rat ESC-specific genes selected from Adherens ons Associated Protein 1 (Ajap1), n 5 (Cldn5), Cdc42 guanine tide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gjb5), Insulin-like growth factor binding n-like 1 (Igfbpl1), Interleukin 36 beta (Il1f8), Interleukin 28 receptor, alpha (Il28ra), ight determination factor 1 (Lefty1), Leukemia inhibitory factor receptor alpha (Lifr), osphatidic acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 8), Caudal type homeobox 2 (Cdx2), Fibronectin type III and ankyrin repeat domains 1 (Fank1), Forkhead box E1 (thyroid transcription factor 2) ), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid ription factor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lef1), Sal-like 3 (Drosophila) (Sall3), SATB homeobox 1 (Satb1), and miR-632.

17. The method of any one of claims 1-16, wherein the medium comprises about 75 U/mL to about 125 U/mL LIF, optionally n the medium comprises about 90 U/mL to about 110 U/mL LIF.

18. The method of claim 17, wherein the medium comprises about 100 U/mL LIF.

19. The method of any one of claims 1-18, wherein the feeder cells comprise a monolayer of mitotically inactivated mouse embryonic fibroblasts (MEFs).

20. The method of any one of claims 1-19, wherein the LIF in the medium is a mouse LIF or has at least 91% sequence identity to SEQ ID NO: 1.

21. The method of any one of claims 1-20, wherein the MEK inhibitor comprises PD0325901 and/or wherein the GSK3 inhibitor comprises CHIR99021.

22. The method of claim 21, wherein the MEK inhibitor is PD0325901 at a concentration of 0.8 µM to about 1.2 µM, and the GSK3 tor is CHIR99021 at a concentration of about 2.5 µM to about 3 µM or 3 µM to about 3.5 µM, ally, wherein the concentration of the MEK inhibitor is about 1 µM, and the concentration of the GSK3 inhibitor is about 3 µM.

23. The method of claim 22, wherein the concentration of LIF in the medium is about 100 U/mL, the MEK inhibitor is PD0325901 at a concentration of about 1 M, and the GSK3 inhibitor is 021 at a tration of about 3 M.

24. The method of any one of claims 1-23, wherein the medium further comprises DMEM/F12 basal medium and Neurobasal medium.

25. The method of claim 24, wherein the medium comprises: 1x 12 basal medium; 1x neurobasal medium; 1% penicillin/streptomycin; 4 mM L-glutamine; 0.1 mM 2-mercaptoethanol; 1x N2 ment; 1x B27 supplement; 100 U/mL LIF; 1  M 901; and 3 M CHIR99021.

26. The method of any one of claims 1-25, wherein the modifying step comprises one or more rounds of electroporation.

27. The method of any one of claims 1-26, wherein the targeted genetic modification is generated via a homologous ination event.

28. The method of any one of claims 1-27, wherein the targeted genetic modification is generated by employing a targeting vector comprising upstream and downstream homology arms flanking an insert polynucleotide, wherein the homology arms correspond to genomic regions within a targeted genomic locus.

29. The method of any one of claims 1-28, wherein the targeted genetic modification is generated using a nuclease agent that generates a single or double strand break at a targeted genomic locus.

30. The method of claim 29, wherein the nuclease agent is a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a meganuclease, or a CRISPR/Cas system.

31. The method of any one of claims 1-30, wherein the modifying step comprises: (i) introducing into the rat ES cells a heterologous polynucleotide comprising a selection marker operably linked to a promoter active in the rat ES cells; and (ii) culturing in vitro the rat ES cells in alternating first and second culture media, n the first e medium comprises an effective amount of a selection agent for a first time period and the second e medium does not comprise the selection agent, and wherein the in vitro culture ions are sufficient to maintain pluripotency, y selecting the rat ES cells having the heterologous polynucleotide stably integrated into the genome.

32. The method of claim 31, wherein the first and second culture media are alternated every 24 hours, and n the selection marker imparts resistance to an antibiotic, optionally wherein the selection marker comprises one or more of neomycin phosphotransferase (neor), ycin B phosphotransferase (hygr), puromycin-N- acetyltransferase (puror), and cidin S deaminase (bsrr); and optionally wherein the antibiotic is G418.

33. The method of claim 31 or 32, wherein the selection marker has one or more of the ing characteristics: (a) the selection marker comprises a non-attenuated selection marker gene; (b) the selection marker has an sed activity compared to the wild type selection marker; and (c) the rat ES cells comprise multiple copies of the selection marker stably incorporated into the genome.

34. An in vitro e system for culturing and ining pluripotency of rat ES cells, comprising: (a) a feeder cell layer not modified to express leukemia inhibitory factor (LIF); (b) a medium comprising about 50 U/mL to about 150 U/mL LIF, and a combination of inhibitors consisting of a MEK inhibitor and a GSK3 inhibitor; and (c) a population of one or more rat ES cells comprising a targeted genetic modification, wherein the rat ES cells are capable of transmitting the targeted genetic modification through the germline.

35. The in vitro culture system of claim 34, wherein the rat ES cells: (i) lack expression of c-Myc; and/or (ii) have a normal karyotype; and/or (iii) form spherical, free-floating es in culture.

36. The in vitro culture system of claim 34 or 35, wherein the medium comprises about 75 U/mL to about 125 U/mL LIF, ally wherein the medium comprises about 90 U/mL to about 110 U/mL LIF.

37. The in vitro culture system of claim 36, wherein the medium comprises about 100 U/mL LIF.

38. The in vitro culture system of any one of claims 34-37, wherein the feeder cell layer comprises a monolayer of mitotically inactivated mouse embryonic fibroblasts (MEFs).

39. The in vitro culture system of any one of claims 34-38, wherein the LIF in the medium is a mouse LIF or has at least 91% sequence identity to SEQ ID NO: 1.

40. The in vitro culture system of any one of claims 34-39, wherein the MEK inhibitor ses PD0325901 and/or wherein the GSK3 inhibitor comprises CHIR99021.

41. The in vitro culture system of claim 40, wherein the MEK inhibitor is 901 at a concentration of 0.8 µM to about 1.2 µM, and the GSK3 inhibitor is 021 at a concentration of about 2.5 µM to about 3 µM or 3 µM to about 3.5 µM, optionally, wherein the concentration of the MEK inhibitor is about 1 µM, and the concentration of the GSK3 inhibitor is about 3 µM.

42. The in vitro culture system of claim 41, wherein the concentration of LIF in the medium is about 100 U/mL, the MEK inhibitor is PD0325901 at a concentration of about 1 M, and the GSK3 inhibitor is CHIR99021 at a concentration of about 3 M.

43. The in vitro culture system of any one of claims 34-42, n the medium further comprises DMEM/F12 basal medium and asal medium.

44. The in vitro culture system of claim 43, wherein the medium comprises: 1x DMEM/F12 basal ; 1x neurobasal medium; 1% penicillin/streptomycin; 4 mM L- glutamine; 0.1 mM 2-mercaptoethanol; 1x N2 supplement; 1x B27 supplement; 100 U/mL LIF; 1 µM PD0325901; and 3 µM CHIR99021.

45. The in vitro culture system of any one of claims 34-44, wherein the targeted genetic modification comprises an ion, a on, a knockout, a n, a point mutation, or a combination thereof.

46. The in vitro culture system of claim 45, wherein the targeted genetic modification comprises an insertion of a heterologous polynucleotide into the genome of the rat ES cells.

47. The in vitro culture system of claim 46, wherein the heterologous polynucleotide comprises a selection marker, and wherein the selection marker has one or more of the following characteristics: (a) the selection marker comprises a non-attenuated selection marker gene operably linked to a promoter; (b) the ion marker has increased activity compared to a wild type ion marker; and (c) the rat ES cells comprise multiple copies of the selection marker stably incorporated into the genome.

48. The in vitro culture system of any one of claims 34-47, wherein: (a) the rat ES cells are derived from an ACI rat or derived from a Dark Agouti (DA) rat; and/or (b) the rat ES cells are derived from a morula stage or a blastocyst stage embryo.

49. The in vitro culture system of any one of claims 34-48, n the rat ES cells are male (XY) rat ES cells or female (XX) rat ES cells.

50. The in vitro e system of any one of claims 34-49, wherein the rat ES cells have one or more of the following characteristics: (a) the rat ES cells express at least one pluripotency marker ed from Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, and Utf1; (b) the rat ES cells do not express one or more otency markers selected from c-Myc, Ecat1, and Rexo1; (c) the rat ES cells do not express one or more mesodermal markers selected from Brachyury and Bmpr2; (d) the rat ES cells do not express one or more endodermal markers selected from Gata6, Sox17, and Sox7; (e) the rat ES cells do not express one or more neural markers selected from Nestin and Pax6; (f) the rat ES cells express one or more pluripotency markers selected from Oct-4, Sox2, and alkaline phosphatase; and (g) the rat ES cells express of one or more rat ESC-specific genes selected from Adherens Junctions Associated Protein 1 ), Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent n kinase IV (Camk4), ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gjb5), Insulin-like growth factor binding protein-like 1 (Igfbpl1), Interleukin 36 beta ), Interleukin 28 receptor, alpha (Il28ra), left-right ination factor 1 (Lefty1), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 8), Caudal type homeobox 2 (Cdx2), Fibronectin type III and ankyrin repeat domains 1 (Fank1), Forkhead box E1 (thyroid transcription factor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid transcription factor 2) ), Hairy/enhancer-of-split related with YRPW motif 2 , Lymphoid enhancer-binding factor 1 (Lef1), Sal-like 3 (Drosophila) (Sall3), SATB homeobox 1 (Satb1), and miR-632.