KR101563017B1 - Transgenic chickens with an inactivated endogenous gene locus - Google Patents

Abstract

The present invention relates to techniques for producing transgenic birds derived from prolonged cultures of avian PGCs and transgenic birds derived from prolonged PGC cultures. In some embodiments, the PGC can be transfected with a genetic construct to transform the DNA of the PGC, specifically to introduce a transgene encoding the exogenous protein. When combined with host avian embryos by known procedures, this modified PGC is transferred through the gonads to produce transgenic offspring. The invention encompasses compositions comprising prolonged cultures of PGCs and progeny derived from genetically modified strains thereof. Genetic modification introduced by PGC to achieve gene inactivation may be achieved by random integration of the transgene into the genome, site-specific changes in the genome introduced using the integrase, site specificity for the genome introduced by homozygous recombination But are not limited to, mutations, and conventional mutations introduced into the genome by cleavage of DNA adjacent to another sequence that is a substrate for Lox site or site-specific recombination.

Description

TRANSGENIC CHICKENS WITH AN INACTIVATED ENDOGENOUS GENE LOCUS < RTI ID = 0.0 >

Transgenic animals provide, for example, the potential for continued production of valuable pharmaceutical products such as antibodies. However, producing transgenic animals involves considerable technical barriers that have been overcome only for some species. The ability to introduce a genetic modification encoding an exogenous protein into another species of DNA requires several unique techniques that must be developed for each species. One approach to altering the genetic and physical characteristics of an animal involves introducing the cells into recipient embryos of the animal. These cells can contribute to the tissues of animals born from the recipient embryo and have the ability to contribute to the genome of the transgenic offspring of the resulting animal.

In certain instances, cells may be engineered with an implantable gene, including, for example, DNA encoding an exogenous product, such as a protein or an antibody. The transgene contains a blueprint for protein production and includes sufficient coding and regulatory elements to enable expression of the protein in the tissues of the animal formed by inserting the cells into the recipient embryo. Under some circumstances it is desirable that expression is ubiquitous so that expression can occur in all tissue types. However, for example, under most circumstances where valuable proteins are required, such as the collection of valuable antibodies, expression should be limited to certain specific tissue types that can facilitate the collection of expressed proteins. For example, through the expression of proteins in a broth, proteins can be easily collected simply by collecting the broth and isolating the exogenous protein. In chickens, active antibody production in egg white provides a vehicle of interest for the expression and collection of valuable proteins. In addition, where tissue specific expression is specific to the fallopian tube of chicken, the expression produces antibodies with specific and desirable chemical properties that increase the therapeutic utility of the antibody when used in the treatment of human patients. Thus, one area of particular interest in research and commercial development is genetically engineered chickens capable of selectively expressing antibodies in egg white or egg yolk so as to facilitate the isolation and collection of proteins with desirable chemical properties.

In the case of exogenous antibody production, algae biological systems offer many benefits of efficient farming, rapid growth, and economical production. In addition, avian eggs provide an ideal design for both mass synthesis of antibodies and easy isolation and collection of products. Additionally, the advantages of a transgenic chicken expression system as compared to, for example, vertebrate, plant, or bacterial cell systems, as described below in connection with the present invention, can be readily demonstrated, Can be applied to have unique beneficial chemical properties for the product. The goal of creating transgenic chickens has been added by scientists over the years. The inherent limitations of the size and / or lack of expression of the transgene gene that can be introduced into the DNA of the transgenic animal, although the goal is achieved, for example, in other species such as mice, cows and pigs, Transgenic chickens could not be produced without the use of retroviral technology or direct injection techniques that had gone through. In addition, viral vectors are not well suited for applications requiring site-specific changes to the genome, such as, for example, provided by homologous recombination.

In addition, under some circumstances, an animal's own endogenous gene may interfere with the production of valuable protein resulting from the introduction of a gene construct specifically designed for the expression of a valuable protein. The ideal solution under such circumstances would be to inactivate the endogenous gene of the animal. Unfortunately, transgenic chickens with site-specific strains that inactivate the endogenous locus have not been described, since genetic engineering of chickens is a unique challenge. In addition, the introduction of site specific gene inactivation results in the production of animals that have lost endogenous gene function, and such animals can be crossed with other animals in which complementary and specific genetic modifications have been introduced into their genome. For example, an animal family deficient in a specific gene can be established, which can be combined with an animal containing a specific gene for humans through crosses. In this case, for the production of a protein encoded by a specific animal phenotype production or by the insertion of an endogenous gene, not only endogenous gene inactivation but also genome-modified introduced animal clusters will be generated. There is no such animal presently because viral vectors can not target the endogenous genome site-specifically and have no ability to select integrated events. Thus, viral vectors do not provide a mechanism to activate the endogenous locus.

If the cell culture is sufficiently stable in allowing the large transplant gene to integrate into the genome of the cell or to introduce a site specific change into the genome, the target cell and several The transgene encoding a tissue-specific expression of any protein by different techniques can be transferred to the transgenic organism. The same technique can be used to perpetuate an organism with an inactivated endogenous gene. Whole genomes can be transferred by cell hybridization, by intact chromosomes by microcells, by subchromosomal segments by chromosome-mediated gene transfer, and by kilo-base-range DNA segments by DNA mediated gene transfer (see, Complete chromosomes can be transferred by microcell-mediated chromosomal transfer (MMCT) (Kobbutcher, LA and FH Ruddle, Annu. Rev. Biochem., 50: 533-554, 1981) A specific design for any such transgene or gene inactivation involving an exogenous gene may also be used to screen for an exogenous gene, The nature of the substance, any gene inactivation, and the phenotypic characteristics of the animal should be considered.

Insertion of a transgene capable of inactivating the endogenous locus or allowing tissue specific expression may threaten the pluripotency of the cell unless the transgene is carefully designed. Thus, a suitable cell line should be stable in culture, and when the cell is transfected with a gene complex that is sufficiently large and complex enough to inactivate the gene, or, if desired, contain all the elements required for tissue specificity and high level expression It must be versatile. In the resulting transgenic animal, the transgene may optionally be expressed in a specific tissue type of each individual designed to express the transgene. Depending on the genetic material of the transgene, the transgene may not be expressed in other tissues if the viability of the animal is compromised or the beneficial chemical properties of the resulting protein are impaired.

Chicken primitive germ cells have been genetically modified by using retroviral vectors within a few hours of isolation from embryos at 11-15 steps (Vick et al., (1993) Proc. R. Soc. Lond. B 251, 179-182). However, the resulting modifications are randomly integrated and the size of the transgene is generally limited to less than about 15 kb, usually less than 10 kb, and most commonly less than 8 kb, Can not be formed using this technique, and transitional cells can not be selected to identify site-specific deformation except for random integration. There has been no report on stable gene modification that requires insertion of more than 15 kb of exogenous DNA into the genome of cultured avian PGCs.

Any limitations on the size or site specificity of any DNA-grafting gene or construct that can be stably introduced into an organ PGC cell culture are important constraints on the ability of the genome of a PGC in a culture to genetically modify it genetically And may ultimately limit the type of genetic modification that can be transferred to the offspring of the recipient embryo through the gonads. For example, it is highly desirable genetic modification to introduce an inactivating vector or extrinsic DNA sequence encoding a protein into the genome of a transgenic chicken. If a large number of such transgenic chickens can be produced, a large amount of valuable protein can be expressed in the chicken and collected in eggs. Bird's eggs provide an ideal repository for biologically active proteins and provide a convenient environment in which proteins can be isolated. Bird animals are also candidates for interest in a wide range of transgenic technologies. However, success was not achieved when all mammalian transgenic techniques were applied to avian species, since there was no cultured cell population that could be transferred to the gonads by the introduction of genetic modification. In a recent paper, Sang et al., "PGCs can be maintained in culture for a prolonged period of time required to identify gene target events without losing their ability to migrate to the germplasm occurring after metastasis, (It is unlikely that the PGCs can be maintained in culture and proliferate for the extended period of time to identify the germicidal targeting events without losing their ability to migrate to the developing gonad after transfer " Prospects for Transgenesis in the Chick , Mechanisms of Development, 121, 1179-1186 (2004)). Thus, to date no genetically transfected PGC has been produced, nor has it been proven to transfer genetic modifications introduced into avian PGCs into live adult animals.

Primordial germ cell (PGC) is a precursor of sperm and egg, and is isolated from somatic tissues in the early stages of development in most animals. In accordance with the present invention, chicken PGC is isolated, cultured, and genetically modified while maintaining its commitment to the gonads of PGC. In addition, PGCs are induced and differentiated into embryonic germ cells (EGCs), which are similar to chicken embryonic stem cells (ESCs) in that they are committed to somatic tissues of PGC. These PGCs are committed to somatic tissues and gonads and provide a unique source of genetic modification of the genome in chickens.

Transgenic animals, particularly mouse production, were important in explaining the gene function of mammals. The traditional approach is to randomly integrate the transgene into the genome, or to target and insert the transgene into a specific locus by homologous recombination.

Random insertion of the transgene has two disadvantages. The first and the main disadvantage is that many genes play a role in providing essential functions at various stages of development and omitting the transcription of these genes frequently results in death of the embryo. Embryo death can be prevented in advance by using site-specific recombinase, for example, Cre-LoxP or Flp-FRT, which confers tissue specificity and gene expression regulated by development stage under the control of a promoter, for example, Cre-LoxP or Flp-FRT. In this case, the gene can be inactivated (termed conditional genetic modification) at discrete time in discrete cells and / or during developmental stages in the same context as other normal animals, using site specific recombination. For example, a Cre-LoxP system can be used to specifically deactivate the insulin receptor gene in? Cells to form insulinotropic defects similar to insulin secretion defects in type 2 diabetes (Kulkarni et al. 1999 Tissue-specific knockout of the insulin receptor in pancreatic beta cells creates an insulin secretory defect similar to that in type 2 diabetes. Cell 96: 329-39). Initially, it was found that embryonic lethality occurs when the unreacted allele of DNA polymerase beta is homozygous. A conditional knockout approach was used to analyze the possible requirements for antigen-receptor gene alignment to deficient DNA polymerase beta in T cells (Gu et al. 1994. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265: 103-106).

Random insertion of the transgene gene, and targeted insertion, both suffered from the inability of transgenic animals to fail to cleave the positive selection cassette from the transgene. The presence of a selective cassette can lead to many problems, for example, gene expression degradation at adjacent loci due to strong transcriptional control elements frequently present in the selection cassette (Lerner et al. 1993 CD3 zeta / et al. 1994. Targeted disruption of the CD3 ecta locus causes high lethality in mice (J et al. : modulation of Oct-1 transcription on the opposite strand EMBO J.13: 1157-65). Positive selection cassettes can be removed by using site-specific recombinase under the control of a tissue-specific promoter.

Cre is a recombinant enzyme that promotes recombination between two LoxP sites, a DNA element consisting of 34 base pairs. Once the two LoxP sites are integrated into the genome in the same orientation, recombination catalyzed by Cre cleaves intervening DNA. The LoxP site may be integrated into the transgene prior to insertion into the genome at random, or the LoxP site may be inserted into the correct position in the genome using the target vector. After cleavage of intervening DNA, adjacent LoxP sites are converted to a single LoxP site. A mutant LoxP site that produces a product that is not well recognized after Cre-cleavage is also available. Flp recombinase, another member of the lambda intergram superfamily among site-specific recombinants, shares the same DNA recombination mechanism as Cre recombinase. Similar to Cre, the Flp recombinase recombines two defined target sites (FRT sites) consisting of 34 base pairs. After cleavage of the intervening DNA, adjacent FRT sites are also converted to a single FRT site.

One of the applications of conditional knockout is to express lethal products in cells that are removed from the precise developmental stage of a particular tissue. For example, Grieshammer et al. (1998 Muscle-specific cell ablation conditional Cre-mediated DNA recombination in transgenic mice leads to massive spinal and cranial motoneuron loss. Dev Biol. 197: 234-47)] Specifically, the Cre-LoxP system was used to express the diphtheria toxin A fragment in muscle cells to study skeletal muscle development in mice. A ligand-regulated form of Cre has also been developed for the purpose of adding a temporary elimination of the Cre-LoxP system so that genetic changes can be precisely induced in the late and / or adult tissues during in vitro or in vivo embryonic development.

Chromosomal rearrangement is a major cause of genetic disease and abortion, which is also associated with cancer progression and maintenance (Ramirez-Solis et al., 1995 Chromosome engineering in mice, Nature 378: 720-4); [Rabbitts et 2001. Mouse models of human chromosomal transposition and approaches to cancer therapy. Blood Cells Mol Dis. 27: 249-59))). Chromosomal translocation usually results in abnormal gene fusion, and as a result, tumor specific mRNAs and proteins are targets of interest for gene therapy. Thus, by using the ability to engineer chromosomal rearrangements using specific breakpoints by the use of site-specific recombinases, a mouse model for human disease has been produced. For example, a translocation corresponding to human rearrangement t (8:21) (q22; q22) and t (9:11) (p22q23) in mice was successfully induced to model a model for acute leukemia [Interchromosomal recombination of Mil and Af9 genes mediated by Cre-LoxP in < RTI ID = 0.0 > mouse development. EMBO Rep. 1: 127-32))). Random chromosome deletion was generated by inserting a LoxP site at a random position in the genome and then expressing Cre recombinase (Zhu et al. 2007. Efficient generation of random chromosome deletion, Biotechniques 42, 572-575).

Conditional genetic modification has become a powerful tool to manipulate gene expression during cell line selection, and analysis of cell fate has contributed to understanding normal development. For example, the Cre-LoxP system has been used to genetically activate phylogenetic tracing in mice in determining the adult fate of engrailed 2-expressing cells originating from midbrain and brain stem stenosis (Zinyk et < RTI ID = 0.0 > 1998. Fate mapping of the mouse midbrain-hindbrain constriction using a site-specific recombination system. Curr Biol. 8: 665-8). This approach included two mice that were crossed. One is a Cre recombinase mouse that expresses Cre under the control of an engraft-2 (En-2) genomic regulatory fragment that directs expression in embryonic midbrain and truncus of the brain. One Cre recombinase mouse is " Quot; and is an indicator / reporter mouse that contains a transgene that provides a permanent record of the recombination event by transforming with a hereditary systemic marker. The indicator mouse system has a LoxP-terminus of a transcription / translation-LoxP-stop cassette that is induced by a regulatory sequence derived from the broadly expressed chicken beta actin gene. When the En2-Cre mouse and the indicator mouse are mated, the double transgenic will carry a copy of each transgene. Recombination between reporter constructs LoxP was achieved only in cells expressing Cre under the En2 regulatory element, truncation at the stop and allowing lacZ expression. Since the Cre-mediated cleavage is cytotoxic, the marked cell and all its offspring have expressed lacZ at a later stage even after Cre is no longer expressed. Thus, staining for LacZ in the brain of adult dual transgenic animals revealed that both cells expressing Cre transiently during the midbrain and hindbrain stenosis were descendants.

Summary of the Invention

The present invention relates to a technique for genetically engineering transgenic chickens and transgenic birds and for producing transgenic chickens containing inactivated endogenous loci generated by homologous integration of targeting constructs into primitive germ cells , ≪ / RTI > long-term PGC cultures. Such transgenic chickens have an integrated transgene gene integrated into the genome of chicken primitive germ cells by homologous recombination resulting in gene inactivation resulting from deletion of at least some of the endogenous locus. The present invention includes stable cultures of primitive germ cells comprising the transgene construct, the transgene (often referred to as a knockout vector, a target vector, a knockout construct, etc.), wherein the endogenous gene inactivation The designed transgene can be stably introduced into the genome of primitive germ cells maintained in culture for a time sufficient for the recombination event to be achieved and selecting the transfected cells.

The present invention also includes primitive germ cells and generated transgenic chickens, including but not limited to site specific deletions of a portion of the gene necessary for endogenous gene expression, whose genome has been modified by inactivation of the endogenous locus do. In all of the above embodiments, the present invention also includes a resulting transgenic chicken, which is produced from a site-specific modification of the endogenous genome. The invention also relates to antibodies produced in chickens having beneficial chemical properties that improve the therapeutic utility of the antibody in certain applications. Antibodies produced in chickens have a unique chemical modification pattern when compared to antibodies produced in vertebrate, plant, or bacterial cell systems, and are useful in the production of antibodies for the purpose of binding toxins to target tissues, When the produced antibody is administered to the patient, the target tissue is treated with increased therapeutic efficacy. In one embodiment, long-term PGC cultures are engineered using genetic constructs specifically designed to introduce genetic modifications into the algae, including insertion of transgene genes that allow expression of the exogenous protein to be tissue-specific. Whether engineering the inactivated loci in the same pluripotent cell or engineered the discrete populations of transgenic chickens containing the inactivated endogenous locus, the engineered process can be used to facilitate the expression of the exogenous protein, Transgenic birds carrying a combination of exogenous DNA encoding protein expression in combination with transgenic chickens having an inactivated endogenous locus for the purpose of crossing with an inserted bird are of unique interest Lt; RTI ID = 0.0 > exogenous < / RTI >

Transgenic chickens with inactivated endogenous loci also provide valuable animal models for unique gene function selection that are valuable for gene expression studies and can not be accomplished without the ability to inactivate selected endogenous loci. Similarly, inactivation of the endogenous chicken locus can occur at specific sites of the endogenous immunoglobulin locus, including the V, D, or J sites, which interfere with immunoglobulin gene rearrangement and inactivate endogenous antibody expression. As a result, one embodiment of the present invention includes transgenic chickens that are endogenous immunoglobulin gene expression resulting from site-specific genetic modification at selected sites of the endogenous chicken immunoglobulin locus, and substantially lack of endogenous immunoglobulin protein production. In a preferred embodiment, the transgene is constructed for targeted inactivation of both light and heavy chains encoding endogenous immunoglobulin production. The transgenic algae of the present invention can also express the transgene-derived antibody in the fallopian tube, and the antibody accumulates in the egg in large amounts. In a preferred embodiment, the exogenous antibody protein is encoded by a human DNA sequence that is expressed under the background of lack of endogenous antibody production, such that the native human antibody is expressed in the chicken fallopian tube in the absence of endogenous avian antibody production, The ability to collect antibodies only from eggs is created.

The present invention relates to an algal community exhibiting tissue specific expression of an antibody, a transgene construct capable of expressing an exogenous antibody, an isolated composition of an antibody produced in chicken and having specifically defined chemical properties, and a method relating to bird production, antibody production , And uses thereof in humans. The present invention utilizes a special technique for producing chimeric or transgenic algae derived from long term primitive cell cultures and long term primordial cell cultures, wherein the genome of PGC is a stable, integrated, exogenous protein expressing transgene Whereby the offspring of the cultured cells will contain a stable integrated gene for transplantation. When introduced into host alveolar embryos by the methods described below, the modified donor cells produce algae expressing the transgene in a selected somatic tissue with specificity of the resulting animal.

The present invention also includes compositions of exogenous proteins expressed in transgenic chickens and having certain desirable chemical properties as compared to vertebrate, plant, or bacterial cell systems. Specifically, these proteins, particularly antibodies, have reduced fucose, galactose, N-acetylneuraminic acid, N-glycollyluraminic acid, and mannose with increased concentration. Antibodies with some or all of these characteristics exhibit increased therapeutic utility when administered to humans. Specifically, such antibody compositions exhibit improved antibody-dependent cellular cytotoxicity (ADCC). Thus, the methods of the invention include using transgenic chickens to improve the therapeutic utility of antibody compositions by expressing the antibodies in transgenic chickens when based on ADCC effects.

The present invention also encompasses transgenic chickens expressing an exogenous antibody in the fallopian tube having beneficial chemical properties as defined herein so that the exogenous antibody can be enriched to a defined amount in the egg white. In one preferred embodiment, the exogenous protein is a human sequence monoclonal antibody encoded by an implantable gene construct introduced into the genome of the transgenic bird. Human monoclonal antibodies that encode a polynucleotide sequence are specifically engineered to be expressed in the fallopian tube and are contained within a graft gene that contains a suitable promoter and a regulatory sequence that promotes tissue specific expression.

The present invention also relates to long-term cultured algae primitive germ cell (PGC) cultures, and several additional inventions made possible by the production of long-term cultures, wherein algae PGCs are allowed to multiply in the culture, PGC cultures can be extended by passage through subculture to extend the viability of the culture to 40 days, 60 days, 80 days, 100 days or more. The PGC of the present invention proliferates in a long-term culture, and when injected into a recipient embryo, produces a gonadal chimera.

The invention also relates to introducing a genetic material into the genome of the PGC to obtain the desired result. In one embodiment, a genetic construct with surrounding HS4 elements can be introduced into the PGC of the invention to ensure that the transgene product is produced. In another embodiment, genetic modification can be performed by using an intergagase that directs construct insertion into the repetitive elements of the chicken genome. In another embodiment, the DNA encoding the selectable marker can be inserted into the region of the chicken genome to prevent production of the gene product.

Although a conditional mutation has been generated in chicken cells, unlike mice in which transgenic cell lines expressing Cre have been produced, expression of Cre recombinase under the control of promoters that are ubiquitous, tissue-specific, Transgenic chicken cell lines were not prepared. In Murine cells, Cre recombinase (Araki et al., 1997 Efficiency of recombination by Cre transient expression in embryonic stem cells: comparison of various promoters. J Biochem 122: 977-82)) and cell permeable Cre recombinase Transient expression of Nat Biotechnol. 19: 929-33) has been used to cleave DNA between the LoxP sites. [Jo et al., 2001. Epigenetic regulation of gene structure and function with a cell-permeable Cre recombinase. However, in the chicken DT40 cell line, the DNA between the LoxP sites could not be removed even when the Cre recombinase was transiently expressed (Fukagawa et al., 1999). Acids Research 27, 1966-1969). Subsequently, by introducing the Cre transgene into the genome of DT40, the DNA sequence between the LoxP and / or mutant LoxP sites could be cleaved (Fukagawa et al. 1999. The chicken HPRT gene: a counter selectable marker for the DT40 [Dhar et al., 2001] DNA repair studies: experimental evidence (Bacillus et al., 2001); [Arakawa et al., 2001 Mutant LoxP vectors for selectable marker recycle and conditional knockouts BMC biotechnology 1, 7-14] In support of chicken DT40 cell line as a unique model. [Environ Pathol Toxicol Oncol 20, 273-83]; [Kanayama et al, 2005 Reversible switching of immunoglobulin hypermutation machinery in a chicken B cell line. Commun. 327,70-75))).

The ability to make conditional mutations in chickens can be helpful. For example, if there is a stop codon at the side of the LoxP site, it is possible to produce a chicken substantially derived from embryonic stem cells using a gene that induces apoptosis under the control of an ubiquitous promoter that is silenced. If this chicken line is crossed with an algal line carrying a gene encoding Cre recombinase under the control of a promoter expressed in the clear zone, the embryo will not occur. When an embryonic stem cell is injected into an embryo that simultaneously expresses a gene that induces apoptosis, the embryo can be substantially derived from embryonic stem cells.

In other applications, the transgene containing a sequence encoding a selectable marker may be located on the LoxP site side. Transgenic birds carrying such transgene genes can be crossed with algae expressing Cre recombinase under the control of promoters expressed in the gonads. Algae produced from such crosses will hatch after the selectable marker is cleaved.

Brief Description of Drawings

Figure 1a: PGC was maintained in culture for 54 days. Note that the cells were unattached and remained round. Arrow symbols indicate several dividing cells visible to the naked eye among the cultures.

Figure 1b: Long term PGC cell cultures were found to be stable when maintained in culture for at least 136 days. These cells were cultured on a nutrient layer of irradiated STO cells.

Figure 2: Gene expression as measured by RT-PCR of germline marker CVH ( Vasa ) and Dazl . Cells were cultured in culture for 32 days. Lane 1 shows that of the aliquot PGC CVH and Dazl, both are expressed. The second sample of lane 2, as measured under the absence of actin, did not contain sufficient mRNA. CES cells were also analyzed; Actin has been expressed, cES cells did not express the CVH, Dazl was expressed only weakly.

Figure 3: Western analysis of samples 13 and 16 of PGC cultures maintained in culture for 166 days. Testis was used as a positive control and liver was used as a negative control. Rabbit anti-chicken CVH IgG was used as the primary antibody.

Figure 4: Analysis of Telomeric Repeat Amplification Protocol (TRAP) at the chromosome end. Different cell extract dilutions of two different PGC cell lines (13 and 16 cell lines) were maintained in the culture for 146 days. The positive control was composed of transformed human kidney cell line 293 and the negative control was a lysis buffer without any template added. In the PGC and positive control lanes, the repeat sequence was visually observed, suggesting that telomerase is present.

Figure 5a: cEG cells derived from PGC were maintained in culture. Figure 5b: Chicken embryonic stem cells. In both types of cells, attention is paid to small cells, large nuclei (gray) and distinct nucleoli.

Figure 6: Southern analysis of the cx-neo transgene gene in primitive germ cell (PGC) cell lines.

FIG. 7: FACS analysis of antibody-stained DT40 cells (negative control clusters), EG cells, ES cells and PGCs against chicken vasa homologue (CVH) and 1B3. DT40, ES and EG cells were negative for both markers, whereas the majority of PGCs were stained for both CVH and 1B3. The cells used were PGC 102; ES 439 and EG 455.

Figure 8: Southern analysis of the HS4-beta-actin-neo transgene gene in two primitive germline PGC cell lines.

Figure 9: Southern analysis of HS4 beta-actin-eGFP-beta-actin-trans transgene gene in primitive germ cell (PGC) cell line TP103. Plasmid control DNA was linearized using NotI. The DNA was digested with KpnI to release the internal fragment. In TP103 and the plasmid, both fragments of the same size were liberated. If the genomic DNA of TP103 is digested with NcoI, MfeI, and SphI, then a larger band should appear than is present in the corresponding lane of the degraded plasmid DNA. No bands were observed in the lanes of TP103 genomic DNA digested with MfeI, which may be the reason that the bands are too large. In lanes representing NcoI and SphI degradants, a fragment substantially larger than the fragment liberated from the plasmid DNA was liberated from the TP103 genomic DNA, suggesting that the transgene was introduced into the genome of the TP103 cell line.

Figure 10: Karyotype of G-09 showing that all chromosomes are diploid. In one copy of GGA 2, the majority of p-cancers have been deleted or displaced to another chromosome. Another copy of CGA 2 is normal. The cell is ZZ (male).

Fig. 11: Wisdom fragment from day 18, stained with DAPI. The presence of GFP - positive cells in the canaliculus was visible.

Figure 12: Panel stained with DAPI shows this section through the canal of E18 testis. GFP-expressing cells were located in the tubules and stained with anti-CVH antibody.

Figure 13: Transgenic offspring generated in step X (EG & K) through step (H & H) from chimeras with PGCs stably transfected with the beta-actin-GFP transgene. As shown in these photographs, all of the tissues showed GFP expression.

14. A tissue derived from a chimera carrying PGCs stably transfected with a β-actin-GFP transgene prepared for histological examination. The blue DAPI staining indicates that the nucleus is present, and the green fluorescence demonstrates that all tissues express the GFP transgene.

Figure 15: Southern blot analysis showing that the transfected PGC cell line, derived from the clone, can contribute to the germline of chimeric chickens and can be differentiated into EG cells. Top panel: Genomic DNA from PGC transfected with HS4 b actin-eGFP-b actin-puro constructs, transfected PGCs to detect internal ( Kpn I) and junctional fragments ( Nco I, Afl II) Three embryos derived from chimeric roosters produced using and EG cells derived from transfected PGC were digested with restriction enzymes. The digested DNA was separated on a 0.7% agarose gel, blotted on a nylon membrane, and probed with a radiolabeled eGFP sequence. The size of the hybridization fragments was the same for PGC, EG cells, and two embryos expressing green fluorescence (GFP + embryos). The third, non-transmembrane embryo (WT embryo) did not show any hybridization. Bottom panel: shows the schematic of the construct, where the position of the restriction site is indicated and the size of the restriction fragment predicted is also shown at the bottom. There were two Kpn I sites, through which a 5.3 kb fragment was formed. Nco I and Afl II perform a cleavage 1 within the construct, whereby the restriction fragment observed is a junction fragment connecting the construct with the genomic DNA located on the side of the insertion site.

16a. Diagram of the random integrated construct used in this study. Two basic types of constructs were used: a selectable marker cassette (drug resistance marker and EGFP) induced by a strong promoter, and a pseudo-construct with two sets of HS4 insulators on the side. The promoter used was mouse PGK, chicken? -Actin,? -Actin + CMV enhancer (CAG) or ERNI. Constitutions were as follows: a drug consisting of a promoter that induced expression of the neo or puro resistant gene (s); a selectable marker cassette (first row); Addition of CAG-EGFP gene (second line); Insulated drug only Selectable markers alone (third row); The same insulated, selectable marker cassette (fourth row) to which the EGFP gene is added; And a CAG-EGFP CAG-neo construct using the LoxP site located on the side of the selectable marker and the monoclonal gene of interest (boxed as shown). Not I was used to linearize the constructs, and then transfection was performed to obtain the same vector shape as presented.

16a. Diagram of the intergla- tion construct used in this study. The attB containing plasmid in which the attB site is added to the HS4- [beta] -actin-puro construct is on the left. The plasmid used to express the intergrase in the cells from the CAG promoter is on the right. Both plasmids were transfected as circular DNA.

17. Alignment of attL sequence and attB obtained from transfected PGC. attB plasmid and a genomic sequence in a PGC clone derived from an intergraft-mediated transfection. The first line is the wild-type attB site, and the center TTG, the normal recombination junction, is underlined. The attL sequence from the intergram mediated insertion of PGC is shown below. The PGC sequence was compared to attB to determine where splicing occurred between the attB on the plasmid and the pseudo attP site in the genome. In the PGC sequence, the attB sequence donated by the plasmid is in lower case, and the attP sequence on the genome is in bold capital letters.

18a. Alignment of the PO41-like repeats from the PGC insertion site and the PO41 common sequence. The PGC side sequences from all clones, which were inserted into the PO41 like repeats, were aligned with each other and with the PO41 common sequence. The first 21 nucleotides are the attB sequences donated by the vector (shown at the top of the alignment), followed by the donor genome side sequence from each clone. Nucleotides sharing at least the cleavage of the sequence are indicated by black boxes.

18b. Sequence of attP and PO41 sequences. The 100 bp attP site was aligned with a repeat of 100 bp of PO41, or approximately 2.5 copies per 41 bp. The intersecting region of the center of the attP, TTG, is shown above.

Figure 19. Targeting of chicken IgL gene.

20a. At the top row is a diagram of the targeting vector for IgL pKO5, the chicken IgL gene. It was designed to replace the 2.3 kb JC region of the IgL gene with a 3.1 kb HS4-ERNI-puro selectable marker flanking the HS4 insulator. The lengths of the two homology arms are 2.3 kb and 6.3 kb. A puro-resistant clone for green fluorescence can be screened through the? -Actin-EGFP gene at the 3 'end, thereby enhancing the targeted clone. The dotted line at the end is the pKO vector skeleton (Stratagene). What is in the middle line is a diagram (including single variable (V), linkage (J) and constant (C) site genes) of the wild type allele of the germline stereotype of the IgL gene. The restriction sites used for the Southern analysis of the targeted clones are presented (S, Sac I; B, Bst EII) and the size of the wild-type fragment is indicated by the double arrows at the bottom thereof. On the bottom line is the structure of the mutant allele in which the J and C regions are deleted and replaced by HS4-ERNI-puro. A restriction map is provided, the size of the mutant fragment is shown at the bottom. The probes used in Southern analysis are located on both sides of the targeting vector and their positions are also indicated. Scale bar = 1 kb.

20b. Clones Four Southern blot analyzes. Four puromycin-resistant clones were analyzed, two of which were not green (clones 1 and 2) and two of which were green (clones 3 and 4). In the left panel, and degradation of genomic DNA from the clone PGC by Sac I, by hybridization to the probe A were analyzed IgL targeting of gene 5 'side. In the right panel, DNA was digested with Bst EII and hybridized with probe B for targeting on the 3 'side of the IgL gene. Clone 2 appeared as a fragment with a predicted size for the targeted clone of heterozygosity.

Figure 21. PCR of ERNI-puro, a marker for the inactivation of immunoglobulin light chain genes in semen derived from GO cocks made with PGC with IgL knockout. Ten ng of genomic DNA prepared from a venous sample was used in a PCR performed on the ERNI-puro selectable marker present in the IgL knockout allele. A primer for the endogenous chicken? -Actin gene was also included from the control group. As a positive control for ERNI-puro PCR, a PGC with an IgL knockout allele was used.

Figure 22: ALDH3A2 expression in BN algae. The panel at the top is the RT-PCR product for ALDH3A2 on RNA isolated from two homozygous BN alga (BN / BN), one heterozygous BN alga (BN / +) and one wild alga PCR. In addition, negative control (-RT-control), positive genome control for RT-response and two negative controls (-control PCR) for PCR reaction are also presented. The 544 bp and 680 bp bands indicate that there is an aldehyde dehydrogenase that does not contain an intron that is not spliced between exon 5 and exon 6, and the mRNA of the aldehyde dehydrogenase containing it. On the bottom panel, the 597 bp band confirms the presence of RNA in all samples. RT-PCR revealed that ADH is expressed in heterozygous BN algae but not in homozygous BN algae, suggesting that transgene insertion has halted transcription of the gene.

Figure 23. Sequence of RT-PCR products from wild-type alleles in the aldehyde dehydrogenase family 3 member A2 transcript. Products A and B are derived from a transcript that does not contain a 136 bp non-spliced intron present between exons 5 and 6, respectively, and transcripts containing it.

Figure 24: Chicken carrying the UbC-LoxP-stop-LoxP-Reaper transgene. A. Southern blot analysis of three UbC-LoxP-stall-LoxP-ripper transgenic cell lines (6-03, 6-51 and 9-51). Genomic DNA samples from G1 algae and UbC-LoxP-stop-LoxP-Ripper vector were digested with SpHI or BclI. The digested DNA was separated on a 0.7% agarose gel, blotted with a nylon membrane, and hybridized with a radiolabeled Ripper specific probe to identify the connected fragment. The size of the hybridized fragment was larger than the vector for genomic DNA, suggesting that the transgene was integrated. B. Schematic showing UbC-LoxP-stop-LoxP-ripper construct. The transgene gene is composed of the UbC-promoter and the LoxP-stop-LoxP-reperfusion gene. The SV40 polyadenylation signal (SV40) and the blasticidin resistant cassette (bsd) were inserted into the 3 'prime of the UbC-LoxP-stop-LoxP-ripple transgene. The 5 ' and 3 ' LTRs were located on the construct side. The position of the restriction site is indicated, and the predicted limit size is also provided.

Figure 25: Chicken carrying the pLenti-ERNI-Cre transgene. 25a. Southern blot analysis of eight ERNI-Cre cell lines. Genomic DNA samples were digested with BglII. The digested DNA was separated on a 0.7% agarose gel, blotted with a nylon membrane, and probed with radiolabeled Cre. The size of the predicted hybridization fragment was 4.6 kb. 25b. Schematic representation of the ERNI-Cre transgene gene. The transgene gene is composed of the ERNI promoter and the Cre transplant gene. The SV40 polyadenylation signal (SV40) and the blasticidin resistant cassette (bsd) were inserted into the 3 'prime of the ERNI-Cre transgene. The 5 ' and 3 ' LTRs were located on the construct side. The location of the BglII restriction site is indicated and the predicted restriction size is also provided.

Figure 26: FACS classification of GFP positive and GFP negative cells from Doc-2 cell line. Two classes of cells were generated by transfecting the Doc-2 cell line with a cyclic plasmid containing the ERNI-Cre transgene expressing Cre recombinase in PGC. GFP negative cells are the results obtained by cleaving a sequence existing between the LoxP sites on the docking site vector carrying the CX-eGFP gene.

Figure 27: Southern analysis of 2 chickens and chickens showing 10,652 bp transgene integration in the Doc-1 PGC cell line. Genomic DNA from two chicks (lanes marked with C1 and C2) derived from crossing DOC1 PGC cell line (lane marked with P) and GO chimera made with DOC1 PGC was digested with BglII or EcoRI. The lysate was fractionated on an agarose gel, transferred to a nylon membrane, and hybridized with the radiolabeled EGFP sequence. In this analysis, a connec- tion fragment containing a docking site vector linked to a genomic sequence flanked at the vector integration site is detected. The size of these junctional fragments depends on the integration site and is a diagnostic agent for angiogenic gene insertion events in PGC. In this case, the BglII fragment is approximately 12 kb and the EcoRI fragment is larger than 12 kb. Fragment sizes were the same in PGC and chicks derived therefrom, indicating that the chicks were derived from PGCs involving DOCl insertion.

Figure 28: Southern blot analysis performed on Cre-mediated recombination of the ripple transgene by 10 different pLenti-ERNI-Cre transgenic chicken lines. 28a. Southern blot analysis of isolated DNA from brain (b) and muscle (m) derived from a double transgenic embryo carrying a copy of the Cre transplant gene and a copy of the LoxP transgene. Genomic DNA was digested with SacI. The digested DNA was separated on a 0.7% agarose gel, blotted with a nylon membrane, and hybridized with a probe consisting of a portion of the lentiviral vector backbone (blasticidin gene and SV40 sequence). The probes hybridize equally to both the full-length LoxP-ripper transgene and the recombinant LoxP-ripper transgene. The band intensity ratio of the transgene (not recombined) versus the recombinant transgene indicates the activity of the Cre cell line. 28b. Schematic representation of Cre-mediated recombination of LoxP-ripper transgene. The full-length LoxP-Ripper graft gene contains a 1.4 kb sequence (referred to as a stop cassette) in which the LoxP site is flanked in the same orientation. Recombination between the two LoxP sites cleaves the 1.4 kb sequence from the chromosome, leaving only a single LoxP site. After cleavage, the size of the recombinant LoxP-ripple transgene is reduced to 1.4 kb. The positions of the probe and SacI restriction sites are indicated and the predicted limiting size is also provided.

Figure 29: Recombination of three different Ripper LoxP cassette transplant genes (6-03, 6-51 and 9-51) by the Cre4 cell line. Figure 29a: Transgenic embryos (R) carrying only one copy of the LoxP-ripper transgene (R) for the three different LoxP-ripper cell lines (6-03, 6-51 and 9-51) Southern blot analysis of a double transgenic embryo (C + R) carrying one copy and one copy of the LoxP-ripper transgene. Genomic DNA was digested with SacI. The digested DNA was separated on a 0.7% agarose gel, blotted with a nylon membrane, and hybridized with a probe consisting of the ripper gene and a portion of the lentiviral vector backbone (blasticidin gene and SV40 sequence). The size of the predicted hybridization fragment was 2.8 kb for the full length (unreconstituted) LoxP-ripper fragment and 1.4 kb for the recombinant LoxP-ripper fragment. 29b. Schematic representation of Cre-mediated recombination of LoxP-ripper transgene. The full-length LoxP-Ripper graft gene contains a 1.4 kb sequence (referred to as a stop cassette) in which the LoxP site is flanked in the same orientation. Recombination between the two LoxP sites cleaves the 1.4 kb sequence from the chromosome, leaving only a single LoxP site. After cleavage, the size of the recombinant LoxP-ripple transgene is reduced to 1.4 kb. The positions of the probe and SacI restriction sites are indicated and the predicted limiting size is also provided.

Figure 30: Southern analysis of Doc 2 cells (lane 2) from which the uncut GFP-positive Doc 2 cell line (lane 1) and the cx-GFP-cx-neo sequence were deleted. Cells were sorted by FACS analysis for green fluorescence expression. Genomic DNA from two cell clusters was prepared, digested with HindIII restriction enzyme, and the DNA hybridized with the radiolabeled sequence from the puromycin resistant gene. A predicted fragment of 5521 bp was present in GFP-positive (uncut cells) cells and a predicted fragment of 1262 bp was present in the truncated cells. These results suggest that the CX-EGFP-CX-neo sequence present between the two LoxP sites in the docking site construct integrated in DOC2 cells through Cre-Lox recombination is deleted.

Figure 31: Diagram of IgL pKO5B targeting vector. The first line shows the structure of the targeting vector IgL pKO5B. The row shows the vector configuration for the 3 'homology site as well as the 5' homology site, including the LoxP and attP sites. The second row shows the relationship of the targeting vector to the chicken wild type IgL allele. The third row shows mutant alleles formed by J and C gene deletion or gene disruption.

32: KO-07 Southern blot analysis showing IgL locus loss in knockout clones. Left panel: Hybridization obtained for the 5 'homology region when DNA from 5 clonal PGC cell lines transfected with IgL pKO5B was digested with SacI and probed with a SacI-BstEII fragment of 0.5 kb. The wild type IgL locus is approximately 10 kb and the mutant fragment containing the targeted deletion is approximately 4 kb. Right panel: Hybridization obtained for 3 ' homology regions with DNA from 5 identical clones. Genomic DNA was digested with BstEII and hybridized with a 3 '1.7 kb Nsil-MfeI fragment, which is also present outside the targeting vector.

Figure 33: Southern blot showing IgL knockout transitions to five of seven chicken embryos (embryos 2,3,4,6 and 7). 0.0 > IgL < / RTI > allele inherited from homozygous, homozygous KO-07 knockout PGCs.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term chicken embryonic stem (cES) cell refers to an ES cell form, which contributes to the somatic tissue in the recipient embryo derived from the clear zone of the embryo of the X stage (E-G & K) ≪ / RTI > CES cells share mouse ES cells and several in vitro features such as SSEA-1 +, EMA-1 + and telomerase +. ES cells have the ability to colonize all somatic tissues.

As used herein, the term primitive germ cell (PGC) refers to a cell that shows PGC form and contributes only to the gonads of the recipient embryo, PGC is derived from whole blood collected from embryo at step 12-17 (H & H) . The PGC phenotype (1) when the gonadal specific genes CVH and Dazl are strongly transcribed in the cell line, (2) when the cells express strongly the CVH protein, (3) when the cells are injected into the recipient embryo at the X- (4) when the cells cause EG cells (see below); or (5) when they are injected into the recipient embryo at step 12-17 (H & H) When injected into an embryo, it can be established by the fact that the cells transduce the PGC genotype through the gonad (see Tajima et al. (1993) Theriogenology 40, 509-519); [Naito et al., Mol Reprod. Dev., 39, 153-161; Naito et al., (1999) J Reprod. Fert., 117, 291-298)).

As used herein, the term chicken embryo reproduction (cEG) cell refers to a cell derived from PGC, which is similar in function to a murine EG cell. The shape of cEG cells is similar to that of cES cells, and cEG cells contribute to somatic tissues when injected as a recipient of the X-phase (E-G & K).

As used herein, the term transgenic means an animal capable of encoding a transgene gene in its somatic and germ cells, and capable of transferring a gene imparted by the transgene to its offspring. The term transgenic also encompasses genetic inactivation, function disruption, termination, and deletion through the precise deletion of a gene by the use of a deletion of a defined gene segment in the endogenous locus, a transgene or targeting construct integrated into the genome of the primitive germline, Or insertion of other artifacts that functionally inactivate the locus through modification of the nonsense sequence, attP site or site-specific gene, as well as the inclusion of an intrinsic gene locus selective and specific gene locus in the endogenous locus, do. The current retroviral technology can not sustain site-specific transformation or selection of transformed cells, and it is possible to sustain long-term PGC cell cultures and to engineer site-specific gene modifications, for example, genetic inactivation The term transgenic excludes the retroviral system.

However, it includes animals that contain site-specific gene modifications that alter the function of the selected gene and produce the desired phenotype from genetic modification. Such transgenic genes and animals derived therefrom are generally referred to as " knockin ". The transgene can insert a deletion of endogenous DNA of at least 10 kb, preferably 10-25 kb, depending on the size or tissue of the gene selected for targeting. In a preferred embodiment, the transgenic bird has no endogenous gene corresponding to an endogenous gene target that is targeted for total or partial deletion or other functional disruption.

Although described in the examples herein as chickens, other bird species, such as quails, turkeys, pheasants, etc., can be substituted for chickens without proper experimentation, can do.

By inserting a DNA construct designed for tissue-specific expression into ES cells in culture, chickens expressing valuable pharmaceutical products such as monoclonal antibodies in chicken egg white can be produced. See PCT US03 / 25270, WO 04/015123 (Zhu et al). An important technique allowed for these animals is the production and maintenance of actual long-term ES cell cultures that are viable long enough for the genotype of the cloned cells to be engineered in culture.

However, unlike ES cells, primordial germ cells (PGC) have been cultured on the basis of only being short-term. Once the incubation period has passed for a few days, these cells lose their ability to contribute only to the gonads. Typically, PGCs maintained in culture using current culture techniques do not proliferate and do not increase. If it does not grow strong, the culture is a "late" culture, and it can not be maintained indefinitely. These end-stage cell cultures degenerate, lose their unique shape of the cells, and return to embryonic (EG) cells. Embryonic germ cells get a different form from PGC, lose the property that they are limited only by the gonads, and acquire the ability to contribute to somatic tissue when injected at the early stage of embryo development. PGC is the only interest because PGC is known to be a stigma of sperm and oocytes in order to introduce the proposed genotype into the gonads of the recipient embryo so that the animal can transfer the desired genotype to future generations.

Long-term PGC cultures, including or without the inclusion of gene inactivation or exogenous DNA insertions, for example, can be used in a variety of ways, such as sustaining valuable genetic characteristics of important chicken breeding lines that are dependent on the poultry and egg production industry It provides important benefits. At present, we have begun special measures to prevent the loss of valuable mating systems through accidents or diseases. For these measures, a number of lineage members should be maintained as longitudinal axes, and such longitudinal axes should be cloned in many parts of the world. Because it is also important to preserve genetic diversity within the crossing line, it is also necessary to keep a large number of valuable animals in reserve. By preserving the genetic characteristics of valuable mating lines among PGC cell cultures other than living preliminary axons, the cost problems of large pre-mating communities can be avoided.

Long-term PGC cultures were prepared by the method described by van de Lavoir, MC, Diamond, J., Leighton, P., Heyer, B., Bradshaw, R., Mather-Love, C, Kerchner, A., Hooi, L., Gessaro, T., Swanberg, S., Delany, M., and Etches, RJ (2006) Germline transmission of genetically modified primordial germ cells. Nature 441, 766-769.

In order to produce genetically engineered chickens using PGC, genetic modification should be introduced into the genotype of PGC, and the rare genomic cells should be isolated, analyzed and introduced into the recipient embryo to form the GO chimera The population of genetically modified cells should be expanded. A wide variety of genetic engineering techniques for targeting cells in culture are well known. One major difficulty, however, is that in order to alter the genotype of PGCs in the culture, the transfected cells grow and proliferate in the culture while at the same time the culture is suitable for introducing transgenic cells, And must remain viable for a period of time suitable for selection. Successfully transformed cells capable of proliferating are capable of producing many cells (e. G., 10 ⁴ to 10 ⁷ cells) within a few days to several weeks after clone induction or nearly clone induction Lt; / RTI > The progenitor cell will be a rare cell with the desired genetic modification. Typically, such cells are modified to accept the well-known techniques (e. G., Lipoic peksyeon or electroporation) with ^10-4 to 10 ^-7 . ≪ / RTI > Therefore, in order to successfully produce PGCs from cultures, sufficient numbers of cells are proliferated and produced to inoculate cells to provide space and nutrition to the cells so that rare genetically modified cells can be selected from the cultures There is a need.

To provide such clusters, the culture conditions must be sufficiently strong such that cells can be grown from 10 ⁴ to 10 ⁷ cell colonies from individual genetically modified cells for use in in vitro gene analysis and chimeric production . These engineered PGCs will only contribute to the neocortex of oocyte or hatching cells (ie sperm and egg) when the animal is matured. In the resulting animal, the whole body tissue will be derived from the recipient embryo, and the gonads will also include the contribution of both the donor cell and the recipient embryo. Because the contribution to gonads is mixed as described above, these animals are also known as "gonadal chimeras ". Depending on the degree of chimeric sex, the offspring of the chimeric sex may be derived from the donor cell or from the recipient embryo.

The gonads in chickens begin as cells entering the newborn lower lobe of the embryo from the blastoderm layer of the X stage (E-G & K) embryo (Kagami et al., (1997) Mol Reprod Dev 48, 501-510); Petite, (2002) J Poultry Sci 39, 205-228)). As the blastodermal layer progresses forward, the pre-primitive germ cells are swept forward in the reproductive half-moon, which can be identified as a large glycogen-loaded cell. The earliest time to identify cells present in the gonads on the basis of these morphological criteria was approximately 8 hours after the start of the culture (see Hamburger and Hamilton, (1951) J Morph 88, 49-92) 4) when the system is used. Primordial germ cells are present from the fourth stage (H & H) until the reproductive half of the month until they migrate through the vascular structure during stages 12-17 (H & H). At this time, primitive germ cells are small clusters of about 200 cells. Primitive germ cells from the vasculature are transferred to the reproductive ridge and are introduced into the ovary or testis as the germline differentiates (Swift, (1914) Am. J. Anat. 15, 483-516); [Meyer, (1964) Dev Biol 10, 154-190; Fujimoto et al. (1976) Anat. Rec 185, 139-154)).

Until now, primordial germ cells in all tested species failed to proliferate in culture for prolonged periods without being differentiated into EG cells. Long-term culture must be performed so that a sufficient number of cells can be produced to introduce genetic modification or inactivation by conventional electroporation or lipofection protocols. Typically, such a protocol requires 10 ⁵ to 10 ⁷ cells, thus assuming that all cell division occurs simultaneously (1) and (2) produces two viable daughter cells, To produce cells, 17-24 doublings should occur. Introduction of genetic modification into the cell genome is a rare event, typically occurring once in 1 x 10 ⁴ to 1 x 10 ⁶ cells. After genetic modification, cells must be able to establish colonies from single cells that carry and / or express genetic modification. Colonies can be analyzed by PCR or Southern analysis to assess the accuracy of the transgene, and then analyzed using 10 ⁵ to 10 ⁷ cells that can provide sufficient numbers of cells to be injected into the recipient embryo in steps 13-15 (H & H) It should be able to expand into a cluster of cells. Thus, an additional 17 to 24 cell divisions are required to produce cell clusters, and a total of 34 to 58 doublings are required to produce genetically modified cell clusters. Assuming that the cell cycle is 24 hours, cultures should be cultured for at least 34 days and generally 58 days to produce genetically modified primitive germ cells for injection into the recipient embryo at step 13-15 (H & H). Subsequently, the injected cells should be capable of colonizing the gonads, capable of forming functional germ cells, and capable of developing into new individuals after fertilization.

PGCs retained in culture as described herein retain their characteristic PGC form while retained in culture. The PGC form can be observed through direct observation and cell growth in the culture can be assessed by a general technique to determine whether the cells proliferated in culture. Proliferative cell cultures are defined as non-terminal, and at the other two points in the latter, a greater number of cells are observed in the culture. It will be observed that the PGCs of the cultures of the present invention may have 1 x 10 ⁵ or more cells in any particular culture, and that this value increases with time. Thus, the present invention encompasses proliferative PGC cultures that include a greater number of cells after a few days, weeks, or months as compared to the initial point in the culture life. Ideally, after the culture is at least 1, and containing, in growth culture for any period of × 10 ⁵ cells can be observed that it has a more number of cells. In addition, PGCs can be observed to be the predominant species in cultures, taking into account the minimal contribution provided by donor cells other than chicken, the proliferative component of the cell culture is substantially free of other chicken-derived cells , Essentially consisting of chicken primordial germ cells.

The culture is also characterized by being able to be propagated by inoculation, wherein the cell sample or cell aliquot from the existing culture can be isolated and will exhibit strong growth even when placed in a new culture medium. By definition, the ability to subculture cell culture indicates that the cell culture grows and proliferates and is non-terminal. In addition, the cells of the present invention demonstrate the ability to form gonadal chimeras after multiple passages and maintain PGC morphology. As described herein, such proliferation is an intrinsic property of any cell culture suitable for stable integration of exogenous DNA sequences.

PGCs can be obtained by any known technique and can be grown under the culture conditions described herein. However, whole blood is removed from the 15-step embryo and placed directly in the culture medium as described below. This approach differs from the other approaches described in the literature (here, it is placed in culture after the PGC processing and separation step). Differential and active growth between PGCs and other cells derived from whole blood that can coexist from the beginning results in the formation of macro PGC clusters in cultures as described herein. Therefore, PGCs directly derived from whole blood can grow in cell cultures with high cell concentration, can be infinitely subcontracted, exhibit strong growth and proliferation, and PGC in the culture is inherently unique growth and proliferation It becomes a sex cell.

One aspect of the present invention is the use of a plurality of, more than three, more than four, five, ten, fifteen, and more than twenty gonad chimeric transgenic animals, all of which have cells derived from the same PGC genetically in their gonads Animals are produced. Another aspect of the present invention is a method of producing chimeric chimeric germline comprising the steps of generating a germline chimeric community with cells derived from PGC that are genetically identical to their germline, A cluster is formed in which the gap is manifested. The age-related disparities are currently out of the available capacity to cultivate primitive germ cells over time and are as high as 190 days without freezing. Thus, the invention provides cells that have the same PGC-derived cells in their gonads and can be categorized by age to any other integral value within 40, 60, 80, 100, 190, And more than two gonadal chimeras. The present invention also relates to the use of a sexually mature gonad chimera having cells derived from PGCs genetically identical to their gonads with the survival of non-terminal PGC cultures, which are used to produce gonadal chimeras, from which additional gonadal chimeras can be produced Of survival.

Because PGCs can be maintained in culture in an extremely stable manner, cells also form long term storage to produce gonadal chimeras with the ability to produce offspring that are defined by the phenotype of PGC maintained in culture It is stored frozen and thawed.

The ability to produce multiple germline chimeras also provides the ability to transfer PGC-derived genotypes to offspring of germline chimeras. Accordingly, the present invention provides a germline chimeric community having an inactivated endogenous locus in the germline, having genetically identical PGC-derived cells, and a progeny chimeric progeny whose genotype and phenotype is entirely determined by the genotype of PGCs grown in the culture , ≪ / RTI > both. It was observed that the knockout phenotype derived from PGC was introduced into the gonads. Thus, the present invention includes the offspring of gonadal chimeras produced by gonadal transfer to genotypes of primitive germ cells containing an inactivated endogenous locus. Thus, the present invention relates to a primitive germ cell culture comprising a PGC consisting of site specific gene inactivation, a germline chimera with said primitive germ cell as part of its germline, and a progeny of a germline chimera with a knockout genotype and phenotype, Includes each survival.

The ratio of the donor-derived PGC to the recipient-derived PGC in the recipient embryo can be altered so that the germline in the PGC-derived chimera can be easily colonized. In the developing chicken and quail embryos, the primitive germline communities can be significantly reduced or eliminated as the primitive germline communities migrate from the reproductive half-moon to the reproductive ridge when exposed to the epidermis (Reynaud (1977a) [Aige-Gil and Simkiss (1991) Res. Vet. Sci., 50 (1981) Arch Anat. Micro. Morph. Exp. 70, 251-258]; [Bile Soc. Zool. Francaise 102, 417-429] , 139-144)). After incubation for 24 to 30 hours, the epidermis is injected into the pyramidal tract and cultured for 50 to 55 hours. When the primitive germ cells are then introduced again into the vascular structure, the germ line is re-proliferated together with the donor-derived primitive germ cells , Followed by production of donor-derived germ cells (Vick et al. (1993) J. Reprod. Fert. 98, 637-641); Bresler et al. (1994) Brit. Poultry Sci. 35 241-247))).

 The method of the present invention comprises the steps of obtaining PGCs from chickens, for example whole blood of 15-step embryos, placing the PGCs in culture, engineerating for the inactivation of endogenous loci, engineering engineered PGCs Proliferating to increase the number thereof and allowing cross-over vaccination, producing gonadal chimeras from organ cultures of engineered PGCs, and genotyping and phenotyping with genetic inactivation in engineered PGCs Thereby obtaining a progeny chimeric progeny. The methods of the present invention also include the steps of generating a stably transfected PGC that includes an inactivated or functionally disrupted endogenous locus by inserting a gene inactivation or gene "knockout" into the PGC population in culture, Selecting cells from the clusters that carry the transgene of interest; injecting genetically modified cells with a stably integrated transgene into the recipient embryo; transferring the embryo to a germline chimera comprising inactivated loci in the germline , Breeding the gonadal chimeras to sexual maturity and crossing the gonadal chimeras so that gene inactivation results in transgenic offspring derived from cultured PGCs. Genetic modifications introduced into the PGC to inactivate the gene include random integration of the transgene into the genome, transgene inserted into the promoter region of the gene, transgene inserted into the repetitive element of the genome, and introduction of the intergagase Specific site-specific changes to the genome introduced by homologous recombination, and other sequences that are substrates for the Lox site or site-specific recombination are introduced into the genome by cutting DNA located on the side But are not limited to, mutations.

As described below, according to the present invention, the chicken PGC cell line is derived from a large, rounded form of blood (FIG. 1), taken from an embryo at 14-16 steps (H & H). These cells are identified by their ability to produce PGC-derived progeny after long-term culture. In addition, the PGC culture expresses the gonadal specific genes Dazl and CVH (FIG. 2), and the CVH protein is produced by the cells in the culture (FIG. 3). PGC in culture expresses telomerase (Figure 4), which is an important phenotype. In addition, PGCs form embryonic reproductive (EG) cells under appropriate culture conditions (Figure 5). In analogy, mouse and human PGCs will be able to form EG cells when treated in a similar manner. Mouse EG cells will contribute to somatic tissue, and chicken EG cells will also contribute to somatic tissue, as indicated by black feather pigmentation in chimeras. Chicken PGC was genetically modified as suggested by Southern analysis (Fig. 6). In this embodiment, the CX promoter is stably integrated into the genome of PGC, which is also used to promote the expression of the gene encoding aminoglycoside phosphotransferase (APH) integrated into the genome of PGC, and genetically modified Is used to confer resistance to neomycin added to the culture medium to screen PGCs.

[Example]

Example  1. Chicken PGC  Induction of culture

2 to 5 μl of blood harvested from the sinus terminalis of the embryo of step 14-17 (H & H) was treated with stem cell factor (SCF; 6 ng / ml or 60 ng / ml), human recombinant fibroblast growth factor ml or 40 ng / ml), 10% fetal bovine serum, and 80% KO-DMEM conditioned media. Preferably 1 to 3 [mu] l was harvested from the vascular structure of the embryo in steps 15-16 (H & H). STO cells irradiated with irradiation in a well of a 96-well plate were seeded at a concentration of 3 x 10 ⁴ cells / cm 2.

10% fetal bovine serum, 1% pen / strep; BRL cells were cultured to saturation in DMEM supplemented with 2 mM glutamine, 1 mM pyruvate, 1X nucleoside, 1X non-essential amino acid and 0.1 mM [beta] -mercaptoethanol and incubated in DMEM containing 5% fetal bovine serum And cultured for 3 days to prepare KO-DMEM conditioned medium. After 24 hours the medium was removed and the medium of the new batch was conditioned for 3 days. This was repeated three times, and the PGC culture medium was prepared by combining the three batches.

After approximately 180 days in culture, one PGC cell line was cultured in a medium consisting of 40% KO-DMEM conditioned medium, 7.5% fetal bovine serum and 2.5% chicken serum. The doubling time of PGC under these conditions was approximately 24-36 hours.

When initiating the culture, the predominant cell type was fetal red blood cells. The predominant cell type within 3 weeks was PGC cells. Two PGC cell lines were derived from 18 cultures starting from individual embryos.

The PGC cell line was cultured for more than 9 months, which remained round and remained unattached (FIGS. 1A & B). After cryopreservation in CO ₂ -independent medium containing 10% serum and 10% DMSO, PGC was thawed successfully.

Example  2. Cultured PGC The CVH  And Dazl ≪ / RTI >

Expression of CVH , the chicken homologue of VASA , a gonad-specific gene in Drosophila, is restricted to cells in the gonad of chicken, which is expressed by approximately 200 cells during the reproductive half of the month (Tsunekawa, N., Naito, M. , Sakai, Y., Nishida, T. & Noce, T. Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells, Development 127, 2741-50. CVH should be expressed for proper function of gonads in males; CVH Loss of function causes infertility in male mice (Tanaka, SS et al., The mouse homolog of Drosophila Vasa is required for the development of male germ cells, Genes Dev 14, 841-53. (2000)). Expression of Dazl can be detected in frogs (Houston, DW & King, ML A critical role for Xdazl, a germ plasm-localized RNA, in the differentiation of primordial germ cells in Xenopus (Development 127, 447-56, 2000) Expression of axolotl DAZL RNA, a marker of germ plasm: widespread maternal RNA and onset of expression in germ cells approaching gonad. ≪ RTI ID = 0.0 > Dev Biol 234, 402-15, 2001), mice (Schrans-Stassen, BH, Saunders, PT, Cooke, HJ & de Rooij, DG Nature of the spermatogenic arrest in Dazl- / - mice. Biol Reprod 65, 771-776, 2001), rat (Hamra, FK et al., Production of transgenic rats by lentiviral transduction of male germ-line stem cells, Proc Natl Acad Sci USA 99, 14931-6, 2002) (Lifschitz-Mercer, B. et al., Absence of RBM expression as a marker of intratubular germ cell neoplasia of the testis. Hum Pathol 31, 1116-1120, 2000). Dazl defects resulted in defects in spermatogenesis in transgenic mice (Reijo, R. et al., Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene, Nat Genet 10, 383- 93, 1995).

After 32 days, the PGCs were washed with PBS, pelleted, and mRNA was isolated from tissue samples using the Oligotex Direct mRNA kit (Qiagen). Subsequently, cDNA was synthesized from 9 mu l of mRNA using superscript RT-PCR (Superscript RT-PCR) system for First-Strand cDNA synthesis (Invitrogen). 2 [mu] l of cDNA was used in the next PCR reaction. The primer sequences derived from the CVH sequence (Accession No. AB004836), the Dazl sequence (Accession No. AY211387), or the [beta] -actin sequence (accession number NM_205518)

Primer V-1 and V-2 were used to amplify the 751 bp fragment from the CVH transcript. The 536 bp fragment was amplified from the Dazl transcript using primers Dazl-1 and Dazl-2. The 597 bp fragment was amplified from the endogenous chicken beta -actin transcript using primers Act-RT-1 and Act-RT-R. PCR was carried out using AmpliTaq Gold (Applied Biosystems) according to the manufacturer's instructions (Fig. 2).

Example  3. PGC The CVH  Express the protein

Proteins were extracted from the newly isolated PGCs using a kit for T-Per tissue protein extraction (Pierce). 1% NP ₄ O; 66 mM EDTA supplemented with 0.4% deoxycholate; Cells were lysed in 10 mM Tris (pH 7.4) to extract proteins from the cells. Samples were developed on a 4-15% Tris-HCL ready gel (Bio-Rad). After transferring to the membrane, Western blotting was performed according to the instructions using Super Signal West Pico Chemiluminescent Substrate kits (Pierce). Rabbit anti-CVH antibody was used as the primary antibody (1: 300 dilution) and HRP-conjugated goat anti-rabbit IgG antibody (Pierce, 1: 100,000) was used as the secondary antibody (FIG.

Example  4. Cultured PGC The Telomerase  Express

Primordial germ cells were pelleted, washed with PBS and frozen at -80 ° C until analysis. Cell extracts were prepared and transformed with Telomeric Repeat Amplification Protocol (TRAP) (Kim, N. et al., Specific association of human telomerase activity with immortal cells and cancer. Science 266, 2011-2014, 1994) using the TRAPeze Telomerase Detection Kit (Serologicals Corporation) according to the manufacturer's instructions (Fig. 4).

Example  5. Embryo reproduction ( EG ) Cells PGC  From the culture To be derived  Can

Chicken EG cells were derived from PGC by allowing cells to attach to the plate, removing FGF, SCF and chicken serum, and culturing the cells under the same conditions as those used for ES cell cultures (van van (2006) Chicken Embryonic Stem Cells: Culture and Chimera Production, Methods in. (2006), pp. Enzymology, in press])). The morphology of cEG cells was very similar to that of cES cells (Fig. 5A, B). When cEG cells were injected into the embryo of stage X (E-G & K), the cells colonized somatic tissues and had the ability to produce chimeras that appeared to be identical to chimeras produced by cES cells, Respectively. Chicken EG cells were observed in both the newly derived transgenic PGC cell line and the clone-derived transgenic PGC cell line. Southern analysis of EG cells derived from GFP-positive PGCs showed that EG cells originated from PGC (Fig. 15).

Example  6. Cultured males PGC The From the rooster  Generate functional germ cells

Male primitive germ cell lines were derived from each Barred Rock embryo. After establishing the cell line, the cells were injected into embryos at 13-15 steps (H & H). As a phenotype, the hatched chicks were similar to White Leghorn. Males were bred until sexual maturity and crossed with bardock hens (Table 1). The offspring showed that the injected PGCs metastasized as gonadal exocytosis. The gonad transfer rate of the cock varied from <1% to 86% (Table 1).

[Table 1]

PGC can also be injected into the embryonic descent of the X-stage. After incubation for 209 days, 1000 or 5000 PGCs were injected into the irradiated embryos. The hatched male chicks were bred until sexual maturity and crossed to test for germline metastasis. Gonadal metastasis was observed in 3 out of 4 roosters tested, ranging in frequency from 0.15 to 0.45%. This suggests that PGC can colonize the gonads when injected prior to intestinal embryo transfer. Gonadal metastasis of male PGC was not observed in 1,625 offspring of 14 female chimeras.

Example  7. Cultured female PGC The From a hen  Generate functional germ cells

Female PGCs from Bardock embryos cultured for 66 days were injected into the white regrowth embryos at 13-16 steps (H & H), all of which were white regal hatching. The hen was raised until sexual maturity and crossed with a bardock rooster. Female PGCs metastasized through female chimeras, with a frequency of 69% or less (Table 2).

[Table 2]

Female PGCs were also injected into the male recipient White Leghorn embryos. Male chimeras were bred until sexual maturity and crossed with bardock hens. In 506 offspring of three roosters tested, there was no germline metastasis of female PGC.

Example  8. PGC from Derived  The offspring are reproductive normal.

Three male and four female non-transgenic PGC-derived offspring were crossed together. Eggs between 53 and 100% of the eggs were fertile (Table 3) and embryo hatching occurred in 79-100% of the fertile eggs (Table 3), suggesting that PGC-derived offspring were reproductive normal .

[Table 3]

Example 9 . Primitive germ cells were isolated from embryos at steps 14-17, which appeared to contribute to gonads (see Examples 1-8). At this point, the PGC circulates in the vascular system. Prior to vascular system formation, PGC was placed in the half-month of reproduction in front of the intrinsic parenchyma. Although PGC precursors during the reproductive half of the month are not well understood, it is generally assumed that PGCs are derived from cells in the transparent zone of the embryo of the X phase (Eyal-Giladi and Kochav) (Petit, JN 2002. The Avian germline and Journal of Poultry Science 39, 205-228). While PGC was in the X-stage embryo, it could not be confirmed using the classic morphological criteria used to identify him during the reproductive half of the month. Surprisingly, the placement of the dispersed cells derived from the X-stage bud-lock embryo resulted in PGCs, which appeared to contribute to the gonads. The inventors have demonstrated the above principle by individually collecting the blastoderm, pulverizing it in a Pasteur pipette and mechanically dispersing it. Cells were washed and plated in 48-well plates containing the medium described in Example 1 and pre-irradiated with BRL. After seeding, the culture was inoculated for the first 6-10 days. Subsequently, the mice were inoculated subj ectively according to PGC concentration. Two male cell lines (PGC-A12 and PGC-B11) were established and cultured for 45 and 36 days, respectively, and then injected into the recipient embryos as described in Example 6. Five male chimeras were produced from each cell line. As shown in Table 4, the Bardock phenotype metastasized through the gonads in three out of 10 males, demonstrating that cells intended to be functional PGCs can be cultured in the medium provided.

[Table 4]

Example  10. PGC Of neomycin and To puromycin  Sensitivity to

The sensitivity of PGC to puromycin and neomycin was measured to establish the concentrations of puromycin and neomycin necessary to allow cells expressing antibiotic resistance to grow under the control of the CX-promoter, which is strongly expressed in all tissues. It has been demonstrated in this experiment that neomycin at a concentration of 300 [mu] g / ml is required for 10 days to remove all of the non-transfected cells. Puromycin at a concentration of 0.5 μg / ml was sufficient to remove PGC within 7-10 days.

Example  11. PGC Genetic modification of

20 linear NotI cx-neo transgene of micrograms (20 ㎕) (see Fig. 6) was added to a 5.8 × 10 ⁶ of PGC clusters cultured for 167 days. Cells and DNA were resuspended in 800 [mu] l of electroporation buffer and 8 square wave pulses of 672 volts and a duration of 100 [mu] sec were added. After 10 minutes, the cells were resuspended in culture medium and aliquoted into 24-well plates. Two days after electroporation, the cells expressing the cx-neo transplantation gene were selected by adding 300 μg neomycin per 1 ml of medium. Cells were continuously selected for 19 days. After 19 days, cell sorting was discontinued and the cells were expanded for analysis. In addition, the ratio of PGC was maintained at 300 ug / ml for 31 days, demonstrating that PGC is functionally resistant to antibiotics.

Referring to Figure 6, in the case of the plasmid control, the cx-neo plasmid DNA was linearly aligned with NotI and then digested with EcoRI or BamHI to obtain a fragment (5 kb) slightly smaller than the intact plasmid by HindIII digestion. Degradation with StyI or NcoI liberated an internal fragment of approximately 2 kb of the cx-neo plasmid. By digestion with EcoRI and KpnI, a larger internal fragment of approximately 2.6 kb was liberated. Digestion of the genomic DNA from the PGC cell line with EcoRI, BamHI and HindIII resulted in a band greater than 6 kb, indicating that the cx-neo transgene gene was introduced into the PGC genome. Internal fragments expressed in plasmid DNA after digestion with StyI, NcoI and EcoRI together with KpnI were also present in genomic DNA derived from PGC suggesting that the cx-neo graft gene was unmodified and integrated into the PGC genome. By using conventional transgene construction techniques, for example, additional elements such as regulatory elements, tissue-specific promoters, and exogenous DNA encoding the exemplified proteins can be introduced.

As mentioned above, transgenic animals have been proven in only a few species by transgenic transformation in PGCs. Similar genetic manipulation can be achieved in chicken PGC with reference to genetic manipulation made using ES cells in mice. It is well known in mice that chromosomes are transferred to embryonic stem (mES) cells after using homologous recombination separately to produce chimeric and transgenic offspring. A powerful technique for site-specific homologous recombination or gene targeting has been developed (see Thomas, KR and MR Capecchi, Cell 51: 503-512, 1987; Waldman, AS, Crit. Rev. ONcol. Hematol. 12 : 49-64, 1992)). (Jakobovits, A., Curr. Biol. 4: 761-763, 1994) and the Cre-LoxP system technique to engineer the gene and transfer it to mES cells to produce a stable gene chimera (Ramirez-Solis, R. et al., Nature 378: 720-724, 1995), and the like, (U.S. Patent Nos. 4,959,317; 6,130,364; 6,130,364; 6,091,001; and 5,985,614)) can be used.

The genome of primitive germ cells is generally considered to be at rest, and thus the chromatin may be in a highly condensed state. Extensive testing of the conventional electroporation protocol has suggested that a special method is required to introduce genetic modification into the genome of PGC. As described below, the transgene can be encapsulated with an insulator element derived from the chicken beta globin locus to enhance expression. The inclusion of a β-globin insulator element generally produces clones that can be cultured, analyzed, and injected into the recipient embryo.

Conventional promoters used to induce the expression of antibiotic (e.g., neomycin, puromycin, hygromycin, his-D, blasticidin, giosin, and gpt) resistance genes are unilaterally expressed. Typically, the promoter is derived, for example, from a "housekeeping" gene such as β-actin, CMV, or ubiquitin. Although constitutive promoters are useful because they are typically expressed at high levels in all cells, they are not expressed in all tissues throughout the life of the chicken, but continue to be expressed in most tissues. In general, expression should be limited to developmental stages and tissues in which expression is required. For primitive germ cell selection, the period of time required for expression is the period during which the primordial germ cells are present in vitro when antibiotics are present in the medium. Once the cells have been inserted into the embryo, it may be desirable to discontinue the expression of a selectable marker (i.e., an antibiotic resistance gene). The early response to neural induction (ERNI) promoter is used to limit the expression of the antibiotic resistance gene. ERNI is a gene that is selectively expressed in the early stages of development (e.g., step (E-G &amp; K))) and in cultures. Thus, such promoters may be used for the expression of antibiotic resistance genes Lt; / RTI &gt; Because ERNI is only expressed during the early stages of development, genes that confer antibiotic resistance are not expressed in mature animals.

Example  12. Long term PGC  Homogeneity of cell culture

ES, EG, DT40 (chicken B cell line) and PGC were stained with anti-CHV and 1B3 antibody, an antibody against the chicken barbarian homology, to determine the homology of the PGC cultures after long term culture (Halfter, W ., Schurer, B., Hasselhorn, HM, Christ, B., Gimpel, E., Epperlein and, HH, An ovomucin-like protein on the surface of migrating primordial germ cells of the chick and rat. Development 122, 915- 23, 1996). Expression of the CVH antibody is restricted to germ cells, so that the anti-CVH antibody becomes a reliable marker for it. The 1B3 antigen recognizes ovomucin-like proteins present on the chicken PGC surface during migration of the ovomucin-like protein and during colonization of the gonad.

The cells were washed in CMF / 2% FBS, fixed in 4% paraformaldehyde for 5 minutes and washed again. The cell aliquots stained for Vasa were permeabilized using 0.1% Triton X-100 for 1-2 minutes. The primary antibody was added for 20 minutes and the cells were washed twice and incubated for 15 minutes with secondary antibodies (Alexa 488 anti-rabbit IgG against CVH and control, and Alexa 488 anti-rabbit IgM against 1B3) for 15 minutes . As a control, the cell aliquots were stained with secondary antibodies only. After another two further washes, cells were prepared for FACS analysis.

Referring to FIG. 7, DT40, ES and EG cells all showed background when stained with CVH and 1B3 Ab. However, PGC stained more strongly than both CVH and 1B3 antibodies. There is a small amount of unstained PGC, either CVH or 1B3, suggesting that small amounts of cells do not express the PGC phenotype. Two maternal PGC cell lines and four transfected cell lines (G-09, P84, P97 / 6 and P97 / 33) derived from PGC13 maternal cell lines were tested against Vasa and 1B3 antibodies (PGC13 and 102). All showed the same pattern, suggesting that various PGC cultures contained the same high percentage of cells expressing the PGC phenotype.

Example  13: Genetic modification of primitive germ cells

When electroporated with a cyclic CX-GFP plasmid, the transient transfection rate of PGC varied between 1-30%. By using eight quasi-wave pulses of 100 μsec and 800 V, we obtained a PGC cell line carrying the CX-neo construct, which was designated G-09. Please refer to Fig. Construct integration was assessed using Southern blot analysis. However, isolating the stably transfected cell line was not a repetitive event. In 37 transfection experiments using both square and exponential decay pulses, except G-09, stable transfection of PGC was not achieved after electroporation of 17 x 10 ⁷ PGCs using a linear construct . In each of these experiments, the number of PGCs varied from 1 x 10 ⁶ to 10 x 10 ⁶ . The following promoters have been extensively used in ES cell studies in mice, chickens and humans: the CX promoter, also called CAG (including the beta-actin promoter with the CMV enhancer) (Niwa, H., Yamamura, K , and Miyazaki, J., Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193-9.1991), MC1 promoter and Ubc promoter. Neither of these promoters increased the transfection rate. Selectable markers could be expressed and insulators were used with integrated constructs to allow genetically expressed cells to induce clones.

The insulator is a DNA sequence that separates the active chromatin domain from the inactive chromatin domain and isolates the gene from the activation effect of the neighboring enhancer or from the silencing effect of the nearby condensed chromatin. In chickens, the characteristic of the 5'HS4 insulator located 5 'of the β-globin locus was well characterized by Felsenfeld and his colleagues (Burgess-Beusse, B., Farrell, C, Gaszner, M. , Litt, M., Mutskov, V., Recillas-Targa, F., Simpson, M., West, A., and Felsenfeld, G. (2002). The insulation of genes from external enhancers and silencing chromatin. Proc. Natl. Acad . Sci . USA 99 Suppl. 4, 16433-7). These insulators protect the β-globin locus from constitutively-condensed upstream regions of the chromatin. The present inventors assembled a transgenic gene with a chicken? -Actin promoter inducing neomycin resistance using a chicken? -Globin 5'HS4 sequence as an insulator of both 5 'and 3' of the chicken? -Actin-neo cassette .

The 250 bp center sequence of the hypervariable region 4 from the chicken β-globin locus was PCR amplified using the following promoter sets:

The PCR product was cloned into pGEM-T and sequenced. HS4 in the pGEM clone was digested with BamHI and BglII to free the insert and digested with BglII to linearize the vector to serially duplicate the HS4 site. The HS4 fragment was ligated into a vector containing the HS4 insulator copy. Clones were screened and clones with two copies of HS4 in the same orientation were screened. This was named 2X HS4.

Example  14: HS4  β- Actin - neo Mass screening using

Β-actin neo was obtained from Buerstedde (clone 574) and transferred to pBluescript. Subsequently, 2X HS4 was cloned at both the 5 ' and 3 ' ends of [beta] -actin neo to produce HS4- [beta] -actin neo. Transfection was performed 8 times using the construct. For each transfection, 5 x 10 ⁶ PGCs were resuspended in 400 μl electroporation buffer (Specialty Media) and 20 μg of linear DNA was added. (ED) (Exponential Decay) pulse (200 V, 900-1100 μF together) or 8 square wave (SW) pulses (250-350 V, 100 μsec) were provided. After transfection, cells were grown for several days and neomycin screening (300 [mu] g / ml) was added. Each transfected product was grown as a grass. Resistant cells were isolated from 5 of the 8 transfectants.

Southern analysis was performed on two transfected cell pools (Figure 8). 2 쨉 g of genomic DNA derived from PGC cell lines P84 and P85, and 20 pg of plasmid (HS4-β-actin neo) were digested. The lysates were developed on a 0.7% gel, transferred to a nylon membrane overnight by capillary transfer in 10X SSC, and probed with radiolabeled neo gene sequences in Rapid Hyb (Amersham) for 2 hours. After washing, the blot was exposed to film overnight at -80 占 폚. Referring to FIG. 7, lane 1 is P84, lane 2 is P85, and lane 3 is a plasmid. For the plasmid control, NotI was used to linearize the HS4- [beta] -actin-neo plasmid DNA. To obtain an internal fragment of 2.3 Kb, the PGC DNA and the linearized plasmid were digested with BamHI. P84 and P85 both showed internal fragments of 2.3 Kb in size. A larger internal fragment of approximately 2.6 Kb was digested with HindIII to free it. In addition, the internal fragment was present in both P84 and P85 digests. When genomic DNA of P84 and P85 were digested with EcoRI and BglII, if the transgene was integrated into the genome, a band of greater than 2.9 Kb would have to appear. No concatenated fragments were observed in P84, suggesting that P84 is a composite of several different clones. In P85, a 4.5-5 kb junction fragment was present in the EcoRI lysate, and a 5 Kb ligated fragment was present in the BglII lysate, which incorporated P85 into the genome, and the culture consisted essentially of one clone . This example demonstrates the utility of insulators as a desirable component of constructs for reliable expression of selectable markers in primordial germ cells.

Example  15: genetically modified PGC Induction of clones

The following examples demonstrate that cell lines of genetically modified primitive germ cells can be induced by clones.

First, β-actin-eGFP was prepared. The eGFP gene was freed from CX-eGFP-CX-puro using XmnI and KpnI and the β-actin was released from HS4-β-actin puro using EcoRI and XmnI and the two were ligated with EcoRI and Β-actin EGFP was produced by cloning into p blue script digested with KpnI. Subsequently, β-actin eGFP was liberated (blunted using T4 DNA polymerase) using BamHI and KpnI and cloned into HS4-β-actin puro digested with BglII and EcoRV.

Transfection was performed 5 times using the construct. For each transfection, 5 x 10 ⁶ PGCs were resuspended in 400 μl electroporation buffer (Specialty Media) and 20 μg of linear DNA was added. One ED pulse (150-200 V; 900 μF) or a SW (350 V, 8 pulse, 100 μsec) pulse was provided. After transfection, cells were plated into individual 48 wells, grown for several days, and then selected (0.5 [mu] g / ml). A total of 5 clones were observed in 4 out of 5 transfected plants. One clone TP103 was analyzed by the Southern method (Fig. 9). Referring to Figure 11, the plasmid control DNA was linearized using NotI. The DNA was digested with KpnI to release the internal fragment. In TP103 and the plasmid, both fragments of the same size were liberated. If the genomic DNA of TP103 is digested with NcoI, MfeI, and SphI, then a larger band should appear than is present in the corresponding lane of the degraded plasmid DNA. No bands were observed in the lanes of TP103 genomic DNA digested with MfeI, which may be the reason that the bands are too large. In lanes representing NcoI and SphI degradants, a fragment substantially larger than the fragment liberated from the plasmid DNA was liberated from the TP103 genomic DNA, suggesting that the transgene was introduced into the genome of the TP103 cell line.

HS4 -β- Actin - cigar Induction of clones

First, puro from CX-EGFP-CX-puro (XmnI-EcoRI), β-actin from β-actin neo in pBS (see above) (Sal-XmnI) Beta -actin puro was prepared by ligation. Subsequently, β-actin puro digested with BamHI was ligated with 2 × HS4 vector treated with BamHI / SAP to clone β-actin puro with pBS containing two 2 × HS4 copies.

Transfection was carried out three times using the construct. For each transfection, 4-5 × 10 ⁶ PGCs were resuspended in 400 μl electroporation buffer (Specialty Media) and 20 μg of linear DNA was added. One ED pulse (200 V, 900 [mu] F) was provided. After transfection, cells were plated into individual 48 wells, grown for several days, and then selected (0.5 [mu] g / ml). No colonies were observed in the two transfectants. Two colonies were isolated from the third transfected product.

HS4 - cx - eGFP - cx - Puro Induction of clones

Transfection was performed three times using HS4-cx-eGFP-cx-Puro. 5 x 10 &lt; ⁶ &gt; PGCs were resuspended in 400 [mu] l electroporation buffer (Specialty Media) and 20 [mu] g of linear DNA was added. Eight SW pulses (350 V, 100 μsec) were provided for each transfection. After transfection, cells were plated in individual 48 wells, grown for several days, and then puromycin screening (0.5 [mu] g / ml) was added.

cx - neo Induction of clones

The PGC 13 cell line was electroporated using a plasmid carrying the cx-neo selectable marker. After exposure to neomycin, a cell line resistant to neomycin was induced (G-09). The karyotype of the cell line was determined, and all cells showed deletion in p-cancer of chromosome 2 (Table 5 and Fig. 10). The data demonstrate that G-09 can be cloned from PGC with signature deletion in p-arm of chromosome 2.

[Table 5]

Example  16: PGC Selectable in Marker  Tissue-specific expression

The gene ERNI is expressed from the transfrontal phase of the chicken embryo and is an early response gene that responds to a signal from Hensen's node (Streit, A., Berliner, AJ, Papanayotou, C, Sirulnik, A., and Stern, CD (2000). Initiation of neural induction by FGF signaling before gastrulation. Nature 406, 74-8). In addition, ERNI is expressed in chicken ES cells (Acloque, H., Risson, V., Birot, A., Kunita, R., Pain, B., and Samarut, J. a new gene family specifically expressed in chicken embryonic stem cells and early embryo. Mech Dev 103, 79-91). In addition to the unique 5 'and 3' UTR sequences, the ERNI gene (also referred to as cENS-1) has a unique structure in which a single long open reading frame is located at the direct repeatable side of 486 bp. Acloque et al. (Acloque et al. 2001) analyzed other parts of the cDNA sequence for promoter / enhancer activity, and found that 3 'UTR It was found that the unique sequence region plays a role as a promoter. PCR primers were designed to essentially amplify a 822 bp fragment of the 3 'UTR of the ERNI gene as described in (Acloque et al., 2001). The ERNI sequence was amplified and cloned into the neomycin-resistant gene upstream with the SV40 poly A site to generate ERNI-neo (1.8 kb). The 2X HS4 insulator was then cloned on both sides of the ERNI-neo selectable marker cassette.

HS4-Erni-neo. &Lt; / RTI &gt; 5 x 10 &lt; ⁶ &gt; PGCs were resuspended in 400 [mu] l electroporation buffer (Specialty Media) and 20 [mu] g of linear DNA was added. One ED pulse (175 V, 900 μF) was provided for the first transfection and eight SW pulses (100 μsec and 350 V) for the second transfection. After transfection, cells were plated into individual 48 wells, grown for several days, and neomycin screening (300 [mu] g / ml) was added. Five colonies were isolated in the first transfection (ED pulse) and 11 colonies in the second transfection (SW pulse).

The isolation of the stably transfected clone suggests that ERNI is expressed in PGC and can be used as a tissue specific promoter.

Example  17: Transfected PGC Contribution to the gonads of

PGCs were transfected with HS4- [beta] actin-GFP and injected into the vascular structure of embryos in steps 13-15 (H & H). On day 18, gonads were harvested, fixed, incised, stained with CVH antibody and germ cells were identified. Subsequently, the stained sections were analyzed for the presence of GFP-positive cells in the gonads. GFP-positive germ cells were observed in both males (Fig. 11) and female gonads. Examination of the embryonic brain, cardiac muscle, and liver histological specimens revealed only four green cells on one slide. This data demonstrates that although some cultured PGCs are observed at ectopic sites, the majority of cultured PGC preferentially colonize the gonads.

The sections were stained with anti-CVH antibody to determine whether the GFP-positive cells were germ cells. As can be seen in FIG. 12, GFP-positive cells are also stained for CVH proteins suggesting that GFP-positive cells are germ cells.

Referring to FIG. 12, GFP-positive cells are present in the sections, and DAPI / GFP panel shows that GFP-positive cells are located in the canaliculus. When the germ cells were stained with an anti-CVH antibody, they showed a very dense red circle that showed the outline of the cytoplasm of germ cells. The DAPI / CVH panel shows that these cells are located within the tubules. The last panel shows that GFP-positive cells also stain for CVH and that the canaliculus contains CVP-positive germ cells negative for GFP.

Example  18. Genetically modified PGC Gonadal transition of

The following transplantation genes were injected into the vascular structure of embryos in the 13-14-stage (H & H) embryonic PGC transfected with either β-actin-eGFP-β actin-puro or cx-eGFP-cx-puro Respectively. The chicks were hatched, the males were reared until sexual maturity, and crossed with bardock hens to determine the gonadal transference of the transgene. All blackish offspring were of PGC origin and tested for the presence of the transgene (Table 6). The gonad transfer rate (%) was calculated by dividing the number of black chicks by the total number of chicks evaluated by feather color (Table 6).

[Table 6]

Example  19. The transgene gene is inherited by the Mendelian law.

The blackish offspring produced by crossing between a chimeric cock carrying a Bardock PGC genetically modified to contain either beta actin-neo, beta actin-GFP, or cx-GFP was transfected with the presence of the transgene Were analyzed. As shown in Table 7, the transgene is inherited by approximately 50% of the PGC offspring, suggesting Mendel's inheritance pattern.

[Table 7]

Example  20: genetically modified PGC Accompanied by chimera In offspring  Expression of ubiquitous transgene genes

PGC-bearing chimeras with β actin-GFP stably integrated into the genome were crossed with wild-type hens and embryos were evaluated for GFP expression. An example of expression in the embryo is shown in Figure 13, where GFP was expressed in all tissues of transgenic offspring of Genesis 34 (H & H) and below. Tissues were prepared for histological examination using frozen sections in older animals. Tissues derived from the pancreas, skin, lung, brain, ovary (not shown), kidney, bursa (not shown), duodenum, breast, heart, liver, spleen, stomach, And that it is still expressed predominantly in animals after hatching.

Example  21: HS4  Containing transgene is inserted into the promoter region of the chicken genome

To address the issue of whether HS4-containing constructs were preferentially inserted into specific regions of the genome that would prevent silencing and allow selective markers to be expressed, the present inventors have found that transgenic gene insertion sites in transgenic PGC cell lines Respectively. Genomic DNA was extracted from the transfected PGC cell line and digested with a restriction enzyme that does not cleave the transgene, or a restriction enzyme that cleaves one of the HS4 elements. DNA was self ligated and transformed into E. coli . Cells were plated on ampicillin plates to isolate colonies containing the amp gene from plasmids bound to the genomic sequence located on the vector side.

Plasmids were purified from 31 of the HS4-construct transfected PGC cell lines and sequenced. We performed BLAT (UCSC Chicken Genome Browser Gateway) and BLAST (NCBI) searches to map genomic locations for each insert. Surprisingly, 25 of the 31 HS4-containing constructs were inserted into CpG islands, which are commonly observed at the promoter site, particularly at the site of the housekeeping gene. Genes inserted into the CpG island are defined by EST, and most of them (23/25), whether known or new, are associated (Table 8). CpG islands usually extend from hundreds of base pairs upstream of the transcriptional start site through the first exon to the first intron, and insertion occurs at all of these sites. There was no tendency to transcriptional orientation of the vector as compared to the endogenous gene. Many of these genes have been predicted to be expressed in PGC, for example, based on their known function as housekeeping genes such as isocitrate dehydrogenase, aldehyde dehydrogenase, and mitochondrial solute carriers. As defined by the EST, seven of the insertions were made in the new gene. Five of these ESTs were originally cloned from the gonad or PGC library suggesting that these genes could also be expressed in the PGC cell lines of the invention. Three of the gene insertions were made at more distal introns, not CpG islands. Five of the insertions were made at sites that did not contain any obvious genes. Three of these insertions were made at very contiguous locations in the LINE or satellite repeats.

[Table 8]

Example  22: phiC31 Integraze  Efficient integration using

The present inventors also inserted foreign DNA into the chicken genome using the phiC31 integrase system which catalyzes site-specific recombination between the attB site and the attP site. Because recombination between the &lt; RTI ID = 0.0 &gt; phiC31 &lt; / RTI &gt; attB and attP sites is irreversible, the genomic insertion of the circular construct retaining the attB site is stable and is not cyclicized and intercalated even if the integrase is persistent. Frogs (Allen and Weeks 2005 Nature Methods 2, 975-9. Transgenic Xenopus laevis embryos can be generated using phiC31 integrase.), Mice (Olivares et al., 2002 Nature Biotechnology 20, 1124-8. IX levels in mice; Belteki et al 2003 Nature Biotechnology 21,321-4. Site-specific exchange and germline transmission with mouse ES cell expressing phiC31 integrase) and human cells (Groth et al 2000 Proc Natl Acad Sci US A. 97,5995-6000 Specific phage integration in human cell; Thyagarajan et al 2001, Mol Cell Biol. 21,3926-34. In site-specific genomic integration in mammalian cell mediated by phagephiC31 integrase, phiC31 integrase is a non- Lt; RTI ID = 0.0 &gt; attB-containing &lt; / RTI &gt; plasmid to the bacterial attP site recognized by these genomes, Which means that it contains Pseudomonas -attP region. For efficient integration, it was also confirmed that the introduced plasmid should have an attB site rather than an attP site (Belteki et al 2003; Thyagarajan 2001). attB site was added to the insulated HS4 [beta] -actin EGFP [beta] -actin furo (HS4 BGBP) construct to generate attB HS4 BGBP. As shown in the left panel of Figure 16B, the integrase construct used in this experiment was shown to contain an att-B containing plasmid in which the att-B site was added to the HS4 [beta] -actin EGFP [beta] -actin furo construct. The right panel of Fig. 16B shows the plasmid used for expressing integrase from the CAG promoter in the cells. Two types of integrase were made, one with SV40 nuclear localization signal and the other without this signal. attB HS4 BGBP and CAG-integrase plasmid DNA were co-transfected into PGC as a circular plasmid. A colony was formed at a higher increase than the non-integrase, linear HS4 BGBP, with 0.3 colonies per 10 ⁶ cells using 20 μg of linear DNA compared to 5 to 10 colonies per 10 ⁶ cells using only 5 μg of DNA, The number of colonies increased by more than 20 times. The NLS type integrase formed fewer colonies than the non-NLS type. This result implies that integrase can recognize pseudo attP sites in the chicken genome and can be used for efficient and stable transfection in PGC.

Example  23: Integraze  Identification of insertion site for clone

The increased efficiency of stable transfection using integrase and attB-containing plasmids means that the chicken genome contains the pseudo-attP site recognizable by the phiC31 integrase. To demonstrate that the attB HS4 BGBP plasmid is integrated through an integrase mediated reaction, rather than by random cleavage of the vector, Southern blot analysis was performed on genomic DNA obtained from five independent PGC clones, In each case, an intact, full-length transgene was observed (not shown) with a structure consistent with integration through the attB site. In order to further characterize the recombination breakpoint at the nucleotide level, identify the pseudo attP region, and identify the inserted chromosomal region, the genomic insertion site and vector junctions in the 12 integrase PGCs of the present invention were cloned and sequenced Respectively. Plasmid rescue was performed as described above for the non-integrase cell lines. The present inventors have observed that the cloning efficiency of the splice fragment is significantly reduced; The number of E. coli colonies obtained was reduced to an average of 69 colonies per transgene for the non-integrase PGC cell line and to 3.1 colonies per transgene in the integrase mediated PGC cell line. The cause of this result is unclear, but one possible reason may be that it is more difficult to break down the repetitive DNA (see below) that is attached to the integrase clone into a restriction enzyme. Plasmid DNA was purified from colonies and sequenced to determine attL and flanking sequences (see Table 9 below). Since the genomic DNA was digested with the enzyme that cleaves the transgene, only the site genomic DNA adjacent to the amp gene on the vector backbone was confirmed by this method, and the DNA adjacent to the other side of the transgene insertion site was not confirmed.

Figure 17 shows the junction between the attB plasmid and the genomic sequence in a PGC clone derived from integrase mediated transfection. The top line is the wild-type attB site, the core TTC, which is usually the recombination junction, is underlined (SEQ ID NO: 9) and the attL sequence (SEQ ID NO: 10-21) from the integrase mediated insertion. The PGC sequence was compared to attB (SEQ ID NO: 9) to determine where splicing occurs between the attB on the plasmid and the pseudo attP site of the genome. In the PGC sequence, the attB sequence provided by the plasmid is in the lower case, and the genomic pseudo attP sequence is in the upper case and is shown in bold.

It has been found in each case that the attL sequence, which constitutes about half of the attB sequence on the plasmid, is linked to the pseudo attP site in the genome, which means that the integrase mediates the recombination reaction. Recombination of the plasmid generated attB with the genome was not accurate and generally did not occur in the core TTG nucleotide of attB. BLAT and BLAST searches map the genomic location of each insert. Surprisingly, 7 insertions of 11 insertions that can be mapped occurred in the repetitive DNA sequence. Using the RepeatMasker Web Server (Institute for Systems Biology) and the ClustalW sequence alignment, iterative parts were analyzed and confirmed to be able to classify these sequences into previously identified P041 repeats (Wicker et al). Figure 18A shows the alignment result of the P041 common sequence (SEQ ID NO: 29) and the PO41-like sequence derived from the PGC insertion site. The PGC recession sequences of all clones inserted in P041-like repeats were aligned with the P041 consensus sequence (Wicker et al. 2004) and with each other. The first 20 nucleotides were the attB sequence provided by the vector (as indicated in the alignment results above), followed by the genomic correspondence sequence from each clone. Nucleotides that share more than half of the sequence are indicated by black boxes. Sequence alignment results indicate that two of the PGC cell lines (2-47 SEQ ID NO: 23 and 18-5-36-2 SEQ ID NO: 22) retain the attB HS4 BGBP insert in the same genomic region. These two inserts are independent, as confirmed by the fact that the nucleotide sequence of the attB-pseudo attP junction is different between the two inserts. The other sequences (18-3-12 SEQ ID NO: 24) were identical to the first two except for the 20 bp insert. None of the P041 repeats identified in the inserts were correct copies of the common sequence, and the sequence homology ratio between the PO41 common sequence and the insertion site repeats was 47% (18-3-43) to 77% (1 -30). As shown in Figure 18B, alignment of 100 bp of attP with 100 bp of the common repeat of PO41 revealed sequence identity of about 46%, which is higher than that observed for pseudo attP sites in human cells (Thyagarajan 2001 ). The P041 repeats are considered to have 259 positions in the genome, each consisting of a 41 bp repeat of several kilobases (Wicker et al). Some of the flanking genomic fragments were in the 10-12 kb range; Sequencing of the two ends of these fragments revealed that both were repetitive, which means that the overall length of the repeat is large. Thus, the PO41 sequence is a large, preferred target for the phiC31 integrase in the chicken genome.

The remaining 4 integrase-mediated inserts were in a unique DNA sequence. One of these sequences (19-1-1 SEQ ID NO: 12) was present in the integrated region of the unique sequence on chromosome 21 and the other (1-41 SEQ ID NO: 10) was located on chromosome 5 of the W1 tumor cell line Was inserted into the promoter region of the chicken orthologue. One sequence (5-7, SEQ ID NO: 11) was present on chromosome 1 at multiple positions, representing a local gene family or a low copy number repeat. One sequence (18-4-11 SEQ ID NO: 13) did not match the sequence found on the chicken genome or the general &quot; non-redundant &quot; database, and thus is considered to be a genomic region that has not yet been sequenced. Finally, one insert (2-38 SEQ ID NO: 21) produced only very short sequences consisting of pseudo attP sites that were not visible in the database.

[Table 9]

Example  23: Identification of transgenic chromosomes

The present inventors have also noted chromosomes where insertions have taken place on cell lines that contain inserts in a unique sequence in which the present inventors are capable of locating. Of the 28 independent insertions of PGC, we observed insertions in 17 different chromosomes of the chicken chromosome 38 chromosomes (Tables 8 and 9). Approximately half of the inserts were present on the macrochromosomes (chromosomes 1-6; 13 insertions) and the other half were present on the microchromosomes (chromosomes 7-38; 14 insertions), with one inserted on the Z chromosome. The rate of insertion into the macro- and micro-chromosomes was proportional to the physical contribution to the genome, which means that there is no bias in the area for integration.

Example  24: gene Targeting

The targeting vector was designed so that the J and C regions of the immunoglobulin light chain gene when inserted into the endogenous locus via homologous recombination were replaced with the HS4 ERNI-furo sorting cassette (Fig. 19). As mentioned above, the ERNI promoter (which drives the puromycin cassette) is specifically expressed in early embryos (Acloque et al, homology) and is expected to produce drug resistant colonies in PGC at a frequency similar to other promoters . 20, the top line is a diagram of the targeting vector for the chicken IgL gene, IgL K05. This vector is designed to substitute the J-C region of the IgL gene with a 3.1 kB HS4 ERNI-furoselection marker (designated I and HS4 insulator). Two homologous cancers were 2.3 and 6.3 kB in length. At the 3 ' end, [beta] -actin EGFP allows the screening of furo-resistant clones for green fluorescence in order to concentrate against the targeted clone. The dotted line at the end is the pKO vector backbone (Stratagene). The middle line is a diagram of the wild type allele of the germline structure of the IgL gene with a single variable (V), linkage (J) and constant (C) region gene. The restriction sites used in the Southern analysis of the target clones (S, SacI; B, BstEII) are shown and the size of the wild-type fragment in double arrowheads is shown below. The lower line shows the structure of the mutant allele in which the J and C regions are deleted and replaced with HS4 ERNI-furo. Restriction enzyme maps are shown and mutant fragment sizes are shown below. The probes used in the Southern analysis are located on both sides of the targeting vector and show their location.

Four clones were isolated from 21 transfectants using a total of 1.05 x 10 ⁸ cells and 210 μg of linear DNA. Two clones in the clones expressed GFP, meaning that they were randomly integrated into the genome and retained the GFP gene. Southern blot analysis of four clones using both side-derived probes of the integrating region revealed that one of the non-green clones (clone 2) was homozygous for the target mutant. Figure 20 shows the results of Southern analysis of 4 puromycin resistant clones. Clones 1 and 2 were not green, while clones 3 and 4 expressed GFP. In the left panel, the PGC clone-derived genomic DNA was enzymatically digested with SacI and hybridized with probe A to target the 5 'side of the IgL gene. In the right panel, DNA was digested with BstE II and hybridized with probe B for targeting on the 3 'side of the IgL gene. Clone 2 showed heterozygosity, a fragment of the expected size for the target clone.

Example  25: J-C Knockout  Have a vector GO chimera

The PGC with the J-C knockout described in Example 24 was injected into the vascular structure of a 13-15-group (H & H) white regrowth recipient embryo. Phenotypically, the hatched chicks resemble white leggings. Males were raised to maturity and abdominal massage was performed to recover semen. The results of PCR analysis using the forward primer ERNI-133F: 5'-TTGCTCAAGCCCCCAGGAATGTCA-3 '(SEQ ID NO: 32) and the reverse primer Puro-8R: 5'-CGAGGCGCACCGTGGGCTTGTA-3' (SEQ ID NO: 33) are shown in FIG. In Figure 21, the expected amplified DNA of 248 bp in size was present in the semen of two or more GO chimeras, indicating that genetically modified primitive germ cells were introduced into the gonads.

Example  26: Aldehyde Dihydrogenase Locus  0- Actin - Neo  insertion

5 × 10 ⁶ cells in 400 μl were electroporated into 20 μg of linear β-actin-neo constructs using an exponential decay of 198 V and 900 μF to grow PGCs derived from parental cell line 15, Cm &lt; 2 &gt; well to obtain a single clone. These cells were grown in the presence of neomycin, neomycin resistant clones were grown and transferred to new wells and propagated. To confirm the stable integration of the transgene, the cells were analyzed by Southern blot analysis and sequenced. As a result, the construct was integrated in the promoter region of the aldehyde dehydrogenase gene, an enzyme involved in the aldehyde metabolism, on chromosome 19 .

PGCs harboring beta -actin-neo constructs were grown and injected into embryos at 13-16 step (H & H) recipient embryos. The embryos were hatched and four roosters were grown to maturity and gonadal transitions were tested. The gonad transfer rates of the cocks were 0, 0, 0.5 and 0.5%, respectively. The heterozygous offspring from one of these roosters were grown and mated by reproductive maturation to obtain homozygous offspring.

[Table 10]

Five hybrid pairs of heterozygous rooster and hen produced a total of 73 chicks and were evaluated for the presence of the BN transgene. A total of 10 chickens (14%) were wild-type, 46 chickens (63%) were heterozygous and 17 chickens (23%) were homozygous (Table 10). This distribution was not significantly different from the 18.25 / 36.5 / 18.25 distribution predicted by Mendel's separation law (Chi-square = 6.55). The ratio of chicks died during hatching was similar among the genotypes. These results indicate that insertion of the BN transgene does not induce a lethal phenotype.

The homozygous rooster was grown to the reproductive maturity stage to test fertility. Five roosters were crossed with wild-type hens and fertilization ability, embryo death and hatching rates were calculated (Table 11). Although the fertility of the two roosters was relatively low, the sperm production of these chickens was poor and therefore the sperm count per sperm injection was low. The feasibility of the two roosters was very good (> 90%), and the fertility of a chicken was moderate. The hatchability of fertilized eggs from all chickens was within normal range. Taken together, these results indicate that the breeding function of BN / BN chickens is normal.

[Table 11]

Since the BNN transgene gene was integrated into the aldehyde dehydrogenase gene and did not affect the survival of homozygous chickens, the present inventors evaluated the aldehyde dehydrogenase message transcription.

MRNA was prepared from Oligotex Direct mRNA Kit (Qiagen) from blood collected from two BN / BN homozygous chickens, one BN / + heterozygous chicken and one wild type chicken (+ / +). For first strand cDNA synthesis, cDNA was synthesized from 5 mL RNA using Thermo-Script RT-PCR system (Invitrogen). 1 mL cDNA was used for subsequent PCR reactions using the following primers:

ALDH3A2-3 AGTGGTCACCGGGGGAGT (SEQ ID NO: 34)

ALDH3A2-4 TCACAGACACAATGGGCAGG (SEQ ID NO: 35)

Actin RT-1 AAC ACC CCA GCC ATG TAT GTA (SEQ ID NO: 36)

Actin RT-2 TTT CAT TGT GCT AGG TGC CA (SEQ ID NO: 37)

The ALDH3A2-3 and A2-4 primers (SEQ ID NOS: 34 and 35) amplified 544 and 680 bp PCR products for the aldehyde dehydrogenase family 3 member A2 transcript. Actin RT-1 and RT-2 primers (SEQ ID NOs: 36 and 37) were used to amplify the 597 bp PCR product for actin transcripts. As shown in FIG. 22, the aldehyde dehydrogenase family 3 A2 transcript was detected in heterozygosity (BN / +) and wild type chicken (+ / +) whereas in homozygous BN chicken (BN / BN) , Suggesting that the insertion of the beta actin-neo-transgenic gene resulted in an insertional knockout of the aldehyde dehydrogenase-3 gene without the morphological phenotype.

Verification that the primers amplified the aldehyde dehydrogenase third family member A2 transcript was performed by sequencing the 544 bp and 680 bp PCR products. The 544 bp product is globally included in the 680 bp PCR product, which also contains a 136 bp misplating intron between exons 5 and 6 (Figure 23). These sequences were the same as those of the published chicken genome.

Example  27: Unknown EST As a result, Actin - gfp -b? - Actin - Furo's  insertion

5 × 10 ⁶ cells were grown by electroporation with 20 μg of the linear β-actin-neo construct and plated in 48-1 cm 2 wells to obtain a single clone. These cells were grown in the presence of neomycin to grow neomycin resistant clones. The resistant clones were transferred to new wells and propagated. The cells were subjected to Southern blot analysis to confirm stable integration of the transgene. Sequence analysis confirmed that the construct was integrated into chromosome 8 with a novel gene (EST C0769951).

The PGC construct was grown and injected into the vascular structure of 13-16 (H) H recipient embryos. The embryos were hatched and eight roosters were grown to maturity and tested for gonadal metastasis. The gonad transfer rates of the rooster were 0, 1, 11, 12, 13, 16, 28 and 92%, respectively. The heterozygous offspring from these roosters were grown and mated to reproductive maturity, resulting in homozygous offspring (Table 12).

[Table 12]

The heterozygous rooster and hen mating pairs produced a total of 298 chicks and evaluated for the presence of BGBP transgene. A total of 90 chicks (30%) were wild type, 128 chicks (46%) were heterozygous and 80 chickens (25%) were homozygous. These results are consistent with the 25% wild type, 50% heterozygous and 25% homozygous offspring predictions, which implies that the transgene gene was inherited by the Mendelian law. Most homozygous offspring died at the time of hatching or hatching, and 95% of all chicks died at 6 weeks of age. This result implies that insertion of the BGBP transgene gene resulted in a functional knockout of the gene essential for survival.

To overcome this problem, it is advantageous to insert the transgene at a predetermined position rather than an uncontrolled random insertion. The ability to insert a transgene at a site in the genome has a stronger advantage than random insertion. The transgene inserted for the purpose of overexpression of the protein product is known to be able to express at a high level and can be inserted at a position that has not undergone silencing by heterolytic chromatin erosion. It is also expected that the insertion of the transgene will not cause any deleterious effects on the animal or cell line, which is heterozygous or homozygous. Thus, there is no need to screen a large number of different random inserts to find that expression levels are high and have not destroyed important endogenous genes.

Example  28. Conditional Apoptosis -Induced gene ( Reaper ) Transgenic  Formation of algae

Reaper  Design of transgene gene

Reaper transgene (LoxP-stop-LoxP-Reaper construct) was constructed as follows. D. melanocortin the requester (D. melanogaster), with embryo poly (A) + RNA and F1 REAPER (CAC AAC CAG AAA GTG AAC GA SEQ ID NO: 38) and F2 REAPER (TGT TTG AAT ACA AAA TGA TGC) primers SEQ ID NO: 39 Reaper cDNA was cloned by RT-PCT. Reaper cDNA was inserted into the RI site of the CX-framework to form the CX-Reaper construct. The KpnI site was inserted into the Reaper cDNA 3 'prime of the initiation codon by site directed mutagenesis. A 1.5 kb LoxP-stop-LoxP cassette from pBS302 (Gibco / BRL) was cloned into the KpnI site to form CX-LoxP-stop-LoxP-Reaper. The LoxP-stop-LoxP-Reaper fragment was inserted into the pENTRB2 clone (Invitrogen) using the RI and NotI sites. The LoxP-stop-LoxP-Reaper fragment was then recombined with pLenti6 / UbC / V5-DEST (pLenti Gateway Vector Invitrogen) to form the UbC-LoxP-stop-LoxP-Reaper construct.

ViraPower Lentivirus Expression system  Used Transgenic  Creation of algae

To form lentiviruses harboring the LoxP-stop-LoxP-Reaper transgene gene, the ViraPower lentivirus expression system (Invitrogen) was used to generate high lentiviral titers up to 4.8 x 10 ⁹ cfu / ml. For virus generation, 293T cells were cotransfected with the virapower packaging mixture containing the VSV-G coding plasmid and the UbC-LoxP-stop-LoxP-Reaper construct using lipofectamine. Twenty-four hours after transfection, the virus supernatant was collected and concentrated by centrifugation. HT 1080 cells were transfected with the virus suspension and the virus titer was measured. High transduction and germ line transfer rates were obtained due to the high shear rate.

To infect the chicken embryo with the virus, 1.5 占 퐇 of the concentrated virus solution was injected into the embryonic stem of the X-embryo. After incubation for 3 days at 37.5-38 ° C, the embryos were transferred to a second surrogate egg shell and incubated until hatching at 37.5-38 ° C and 50% humidity. A total of 398 embryos were infected with the virus carrying the UbC-LoxP-stop-LoxP-Reaper graft gene. A total of 155 birds were hatching, and the presence and sex of transplanted genes were analyzed by PCR on DNA isolated from crested tissue. Thirteen male chicks were positive for the UbC-LoxP-stop-LoxP-Reaper transgene. Ten of them were positive for UbC-LoxP-stop-LoxP-Reaper transgene by PCR in DNA isolated from semen. Three males (6-03, 6-51 and 9-51 GO primordial males) transferred the transgene to the next generation. 6-03, 6-51, and 9-51 were 0.32%, 0.26%, and 0.16%, respectively (Table 13).

[Table 13]

6-03, 6-51 and 9-51 Frequency of gonadal transfer to GO rooster

The GO males (6-03, 6-51 and 9-51, with different inserts) were bred to breed and their G1 offspring were analyzed for the presence of the transgene and the integration site of the transgene. Genomic DNA from each alga was analyzed by Southern blot analysis. Genome sample and UbC-LoxP-stop-LoxP-Reaper vector (control group) were digested with SphI or BcII. The digested DNA was separated on a 0.7% agarose gel, blotted with a nylon membrane, and probed with a radioactive Reaper-specific probe to identify the connecting fragment. As shown in Figure 24, the size of the hybridization genomic fragment is larger than the control, indicating that the transgene is integrated. Hybridization genomic fragments for species 6-03, 6-51 and 9-51 differ in size, indicating that 6-03, 6-51 and 9-51 are independent species.

Example  29. Cre - ReComm Pinina  Holding Chicken Formation

Cre  Design and assembly of transgene genes

In order to express Cre recombinase from chicken, a transgene was constructed in which the Cre gene was placed under the transcriptional control of the chicken ERNI promoter. The ERNI gene (also known as cENS-1) is expressed in nervous tissues of early chicken embryos (around the X-phase, which is the stage of the egg just when the embryo is an undifferentiated version of the cell before embryogenesis). Thus, the Cre transplant gene is designed to be expressed in early embryos and promotes the recombination of the LoxP-Reaper transgene gene or other LoxP-containing transgene genes located within the genome. Because Cre is expressed at an early stage, the resulting chicken has a recombinant transgene in every cell layer and every cell in the body.

A lentiviral vector method was used to introduce the Cre transplant gene into the chicken gonads. The lentiviral transgene gene was constructed based on the Invitrogen pLenti6-V5 Dest lentiviral vector. The lentiviral vector component of pLenti6-V5 Dest was combined with the ERNI-Cre gene to generate the pLenti-ERNI-Cre construct. Lentiviruses were generated and used to infect early embryos and were stably integrated into the genome. Approximately 20 transgenic starting birds carrying the pLenti-ERNI-Cre transgene were generated.

The chicken ERNI promoter was PCR amplified using the following primers:

ERNI -738: 5'-ATGCGTCGACGTGGATGTTTATTAGGAAGC-3 'SEQ ID NO: 40

ERNI +83: 5'-ATGCGCTAGCTGGCAGAGAACCCCT-3 'SEQ ID NO: 41

The 822 bp PCR product was cloned into pGEM T-easy (Promega) and sequenced. The ERNI promoter was then digested with SacII (hereinafter blotted with T4 DNA polymerase) and SpeI and released from the vector. Clal (blunt-ended with T4 DNA polymerase) and SpeI to remove the CMV promoter from the lentiviral vector pLenti6 V5-Dest (Invitrogen). The ERNI promoter was then ligated to the pLenti6V5-Dest lentivirus vector backbone, and the CMV promoter inside thereof was replaced with the ERNI promoter to obtain pLenti-ERNI.

The Cre gene was PCR amplified using a convenient restriction site for cloning (BglII at the 5 'end and EcoRI at the 3' end) with the following primers and the SV40 nucleotide sequence on the N-terminus:

Cre-C: 5'-CCG CCG GAG ATC TTA ATG CCC AAG AAG AAG AGG AAG CTG TCC AAT TTA CTG ACC GTA CAC-3 'SEQ ID NO: 42

Cre-Rl: 5'-TCGAATTCGAATCGCCATCTTCCAGCAGGCG-3 'SEQ ID NO: 43

The 1040 bp PCR product was digested with BglII and EcoRI and gel purified. The shuttle vector pENTR 2B (Invitrogen) was digested with BamHI and EcoRI and the vector backbone was gel purified. The Cre PCR product was ligated to the pENTR2B vector and a clone was obtained. Clones were sequenced to determine if the Cre gene was as expected and whether any mutations were obtained during PCR amplification.

In order to recombine the Cre gene into the pLenti-ERNI construct and place it under transcriptional control of the ERNI promoter, the LR clonazepam reaction (Invitrogen) was performed using the pENTR 2B-Cre clone as the Cre gene source and the pLenti-ERNI vector as the receptor. Thus, the final construct pLenti-ERNI-Cre (8408 bp) was obtained and used to generate lentivirus carrying the ERNI-Cre transgene.

pLenti - ERNI - Cre - having a transgene Transgenic  Creation of algae

PLenti-ERNI-Cre lentivirus was generated from 293FT cells. For transfection with 293FT cells each to generate lentivirus, 8 million 293FT cells of 75% confluency were transfected with 3 ug circular pLenti-ERNI-Cre plasmid DNA using the lipofectamine reagent (Invitrogen) Lt; / RTI &gt; The Virapower packaging mixture (Invitrogen) was used to express the viral proteins necessary to produce lentiviruses in 293 FT cells. Two days after transfection, the cell culture supernatant containing lentivirus was collected, the cell debris was removed by filtration, and the lentiviral particles were concentrated by centrifugation at 48,000 g for 90 minutes. The virus pellet was resuspended at 1/200 of the initial volume of the culture supernatant and frozen at-80 C in 40 [mu] l aliquots.

Lentivirus seed to ^10-4 to ^10-infection activity of each batch of lentiviral seed was determined by serial dilution to ⁸ and added to the HT1080 culture using a 1 ㎕ polybrene on HT1080 cells. After 2 days of lentiviral addition, blasticidin selection (5 ㎍ / ml) was initiated. Because the toxicity of blasticidin caused the cells to die, the culture medium was changed every two days. Ten days after the start of blasticidin selection, colonies were stained with crystal violet, counted, and titer was calculated. 10 &lt; ⁸ &gt; to 2 x 10 &lt; ⁹ &gt; To infect the chicken embryo with the virus, 1.5 占 퐇 of the concentrated virus solution was injected into the embryonic stem of the X-embryo. After incubation for 3 days at 37.5-38 ° C, the embryos were transferred to a second surrogate egg shell and incubated until hatching at 37.5-38 ° C and 50% humidity. A total of 310 embryos were infected with a virus carrying the Erni-Cre transgene. 96 chicks hatched and the presence and sex of transplanted genes were analyzed by PCR on DNA isolated from crested tissue. Eight male chicks were positive for the Erni-Cre transgene. Thirteen males were also positive for the Erni-Cre transgene by PCR in semen-isolated DNA. Four out of six males transferred the Erni-Cre transgene to the next generation. The frequency of germline metastasis in the Erni-Cre GO cock varied from 0.24 to 1.32% (Table 14).

[Table 14]

Erni-Cre G0 Gonadal transfer frequency of rooster

pLenti - ERNI - Cre  Having a transgene Transgenic  Analysis of chicken

Southern blot analysis was used to verify that the pLenti-ERNI-Cre transgene was fully integrated into the chicken genome. The genome DNA derived from chicken, which was previously confirmed by PCR in the presence of Cre transplantation gene, was extracted and digested with an enzyme cleaved at the 5'- and 3'-LTR sequences (BglII) of the virus to allow a full-length transplantation gene to be observed . For analysis, algae with different independent transplant gene inserts were selected. Genomic DNA was digested with BglII enzyme, transferred to nylon membrane, and probed with radioactive Cre gene. Figure 25 shows a representative Southern blot result of 8 ERNI-Cre species. Out of the 11 species tested, 10 species included the predicted 4.6 kb ERNI-Cre transgene bands, indicating that the transgene was fully integrated into the genome. One species has fewer bands, indicating deletion or rearrangement of the transgene.

Example  30. Establishment of a cell line capable of site-specific transgene integration

There are two main methods for inserting exogenous DNA into the host genome at a given location, namely site-specific recombination to a recognition site, such as homologous recombination (gene targeting) or attP. Homologous recombination is inefficient in most vertebrate cell types and generally requires screening multiple clones to identify one or several clones with the desired insert. Site-specific recombination is a high-fidelity, high-efficiency process that can be used to insert foreign DNA into a given location without screening multiple clones. Site-specific insertion will depend on the use of the phiC31 integrase to insert the attB containing construct into the native attP site or the caudate-attP site located in the genome. To use the true attP site located within the genome as a docking site, the attP site should be located at a desired location. The attP site is placed at the desired location in the genome by random insertion or homologous recombination. If random insertion inserts the recognition site into the genome, it must be demonstrated that the important gene is not destroyed at the insertion site. The recognition site located within the genome serves as a "docking site" for insertion of the transgene using phiC31 integrase.

In order to specifically select integrase mediated transgene inserts at the docking site, a selectable marker system is used to select the correct inserts. The docking site is designed so that the attP site is adjacent to a drug screening marker (such as a puromycin resistant gene) without a promoter. Therefore, cells with docking sites are susceptible to drug screening using puromycin. The transgene gene to be inserted into the docking site includes a promoter adjacent to the attB site, but does not include a selectable marker. When the transgene is inserted into the docking site, a promoter is placed upstream of the selectable marker to activate transcription and confer resistance to puromycin. Insertion of the transgene at other positions in the genome does not induce drug resistance, and such inserts are eliminated by drug screening.

The attP docking site construct consists of an attP site positioned adjacent to a drug selectable marker that does not contain a promoter, such as puromycin resistance. Because the puromycin resistance gene is not expressed, another selectable marker such as a beta-actin promoter driving neomycin selectable markers should also be included. The EGFP gene should also be included. On each side of these components, there were two copies of the beta-globin HS4 insulator, insulated from adjacent chromatin. To further remove the EGFP portion and beta -actin neo of the construct, the LoxP site is placed in contact with these components. All of these parts of the construct act as a vehicle for transferring the true attP site to the genome. The order of the DNA components is HS4; attP; A puromycin resistance gene without a promoter; LoxP; beta -actin or CAG promoter; EGFP; beta -actin or CAG promoter; Neomycin resistance gene; HS4; Plasmid backbone (pBluescript). This construct is linearized and transfected with cultured PGCs to obtain drug resistant colonies. These colonies are propagated for further analysis.

In order to know the position of the doping site in the genome, it is important to determine the chromosome insertion site of the docking site construct in each clone. Obtain the flanking genomic DNA, sequence it, and compare it with the chicken genome database. Most clones are found to be inserted into CpG islands, which are regions of the genome that are normally linked to the promoter region of genes, particularly housekeeping and ubiquitous genes. Also, most of the inserts are found to be within the promoter region of the gene, the first exon or the first intron. Thus, a number of inserts are predicted to disrupt the function of these genes (Example 29; see Table 8). These genes are known or predicted genes based on the expression sequence tag (EST) sequence. Preferred cell lines are those that do not appear to destroy the gene, such as DOCl or DOC33.

The CAG-EGFP-CAG-neo portion of the docking site can be deleted by Cre-Lox recombination. After Cre-Lox recombination, all that remains in the docking site is the HS4 insulator, the attP site, and the promoterless puro gene. This reduces the number of exogenous proteins produced in cells and transgenic chickens, which can affect health, particularly when expressed singly from strong promoters such as CAG or beta -actin. Cre-Lox recombination can be carried out in cell cultures by transiently transfecting a docking site clone with a circular Cre-expression vector. After several days, the culture is monitored for loss of EGFP expression induced by cleavage of the CAG-EGFP gene. About 50% of the cells no longer express EGFP, and these cells can be classified by flow cytometry and purified (FIG. 26).

Alternatively, transgenic chickens carrying the docking site construct may be subjected to Cre recombination by crossing with a chicken carrying the ERNI-Cre transgene (Cre4 bird).

If transgenic chickens with docking site integration are made, the resulting homozygous chicken may be healthy and reproductive even though it has an insert in the gene. An example of such a cell line is the TP85 cell line (also referred to as BN; see Example 26), and the insert of the gene encodes the aldehyde dehydrogenase third family member A2 on chromosome 19 of chicken. This construct is an HS4-insulated beta -actin neo transgene and is inserted into the promoter region within about 10 bp of the transcription initiation site of the gene. The avian homozygote for the insert is healthy and fertile.

In some cases, however, the insert can cause adverse effects such as developmental disability, anatomical or physiological defects, infertility, and the like. See Example 27. It is therefore important to demonstrate that randomly inserted docking site inserts do not cause any adverse effects in the animal.

Example  31: 10.5 kb  Over expression DNA &Lt; / RTI &gt;

Doc-1 cell line was HS4; attP; A puromycin resistance gene without a promoter; LoxP; CAG promoter; EGFP; CAG promoter; Neomycin resistance gene; HS4 (see Example 29). The construct was linearized and transfected with cultured PGC to obtain drug resistant colonies. These colonies were propagated for further analysis. The Doc-1 cell line was injected into the receptor embryos and the G0 chimeric chick was hatched. The rooster was sexually matured and the sperm was analyzed by FACS analysis for the presence of GFP positive sperm. Two cows were selected for breeding, and the gonad transfer rate was 3% and 8%. Blood was collected from GFP-positive chicks and analyzed by Southern analysis to confirm the presence of the docking site (Figure 27).

Example  32: Targeted  Insert docking area

In order to avoid placing the true attP site within the CpG isoform of the gene, taxon-phone targeting can be used so that the attP site is located at a predetermined site in the chicken genome. Homologous cancers are prepared by screening genomic regions, high fidelity PCR or genomic cloning in plasmid vectors, and assembling targeting vectors with selectable markers for transfection and screening of PGC clones.

Example  33: Site-specific insertion of cellular host with docking site

For insertion into the docking site, an annular construct containing the attB site is constructed. The attB containing construct is similar to that used in the above examples, but there is an important difference that there is no selectable marker. Instead, a promoter (e.g., an ERNI promoter) is placed adjacent to the attB site such that upon integration into the docking site, the promoter is placed at a position that induces the expression of a selectable marker in the docking site.

The promoter-attB backbone can be used for screening for insertion into a puro docking site with no attP-promoter. The attB construct possesses a tissue specific promoter that induces gene expression that encodes a pharmaceutical protein such as another interesting gene, e.g., an antibody.

The function of the docking site and the integration efficiency within the docking site were tested in a PGC cell line containing the docking site. 5 × 10 ⁶ cells were co-transfected with 0.5 μg of construct containing Erni-attB and 0.5 μg of cyclic construct expressing integrase. After electroporation, the cells were replated in 48-1 cm &lt; ² &gt; wells to obtain a single colony. Colonies were observed in 42 wells of 48 wells.

The amplification of the attL site resulting from the recombination of attP and attB confirmed that the ERNI-attB construct was correctly integrated into the docking site. One primer is in the ERNI sequence, one primer is in the puromycin sequence, and amplification can occur when the ERNI promoter is integrated upstream of the puromycin gene. All positive results generated using the three primer sets are as follows:

ERNI-37F + puro-8R Product size 152 bp

ERNI-133F + puro-8R Product size 248 bp

ERNI-342F + puro-83R Product size 522 bp

Primer sequence:

ERNI-37F ACCACGGCAACGGGAGAGGCTTAT SEQ ID NO: 44

ERNI-133F TTGCTCAAGCCCCCAGGAATGTCA SEQ ID NO: 32

ERNI-342F TGGGCAAAGGCAGAGGAATC SEQ ID NO: 45

puro-8R CGAGGCGCACCGTGGGCTTGTA SEQ ID NO: 33

puro-83R GCGTGGCGGGGTAGTCG SEQ ID NO: 46

Four independent clones obtained by transfecting ERNI-attB with DOC2 cells were tested by PCR, and all four clones displayed the correct size amplification product with all three primer sets. ERNI-133F SEQ ID NO: 32 + puro-8R The PCR product formed by the primer of SEQ ID NO: 33 was cloned and sequenced to confirm that the PCR product was correct. The sequence contained fully predicted partial ERNI and puromycin sequences with the predicted attL sequence (combination attB and attP). Thus, integrase mediated recombination events occur in the correct core nucleotides in the attB and attP sites, confirming that they are genuine.

Example  34: Cre From a chicken embryo LoxP  Effectively recombine sites

Ten Cre cell lines (and one cell line with the rearranged ERNI-Cre transplant gene) with intact pLenti-ERNI-Cre transgene were tested for Cre recombinase activity. Although the ERNI promoter is expected to drive the high expression of Cre recombinase in early embryos, it may be silent if the transgene is integrated into an undesirable region of the genome (this phenomenon is known as a 'locus effect'). Therefore, it is important to measure activity on all Cre recombinase in both Cre cell lines to screen for cell lines or cell lines with the desired activity.

In order to determine the activity of the Cre transplant gene, the ability of Cre to catalyze the recombination of the LoxP-ripple transgene in a double transgenic embryo carrying one copy of the Cre transplant gene and one copy of the LoxP transgene Southern blot analysis. The LoxP-ripper transgene contained a 1.4 kb sequence (referred to as a stop cassette) adjacent to the LoxP site in the same orientation. Recombination between the two LoxP sites cleaves the 1.4 kb sequence from the chromosome leaving a single LoxP site. Subsequently, the intervening sequence disappeared because it was no longer linked to the chromosome. After cleavage, the LoxP-ripple transgene gene was reduced in size by 1.4 kb. A Southern blot assay was developed in which size reduction of the LoxP-ripper transgene gene is used to measure Cre recombinase activity. Upon digestion with the restriction enzyme SacI, a (non-recombined) full-length LoxP-Ripper fragment of approximately 2.8 kb was produced when hybridized to a probe consisting of the ripper gene and a portion of the lentiviral vector backbone (blastididin gene and SV40 sequence) . Upon Cre-mediated recombination and cleavage of the 1.4 kb stop sequence, the Ripper SacI fragment was approximately 1.4 kb in size when hybridized with the same probe. The probe hybridized equally to both the full-length LoxP-ripple transgene and to the recombinant LoxP-lipase transgene by hybridizing to a sequence not affected by Cre recombination.

To calculate the Cre activity expressed by the various pLenti-ERNI-Cre transgenic cell lines, the ratio of the band intensities of the (unreassembled) full-length and recombinant transgene was determined. When Cre was not active, recombinant ripper was not observed or was rarely observed, and only the electric field was observed. When Cre was moderately active, both SacI fragments were observed, indicating that recombination occurs in some cells but not in other cells. When Cre is highly active, only the recombinant ribosomes were observed because the LoxP-ripple transgene was recombined in all cells.

Data summarizing the activity of the Cre cell line is shown in FIG. Cre activity in the 11 cell lines tested varied considerably between cell lines, and in some cases also in cell lines. Only one of the 11 cell lines catalyzed the recombination of the 100% LoxP-ripple transplantation gene (Cre4 cell line). In these cell lines, 100% recombination of all tested embryos (18 embryos out of 18 embryos) was shown. In other cell lines, the recombination rate ranged from about 5% to about 80%.

In some of the above cell lines (Cre1, Cre2, Cre11 and Cre20), the embryo-to-embryonic variability was significant in the recombination rate. For example, Cre11 and Cre20 catalyzed only about 10% recombination in some embryos, but less than 60% recombination in other embryos. For most embryos, brain and skeletal muscle tissues were analyzed, and recombination rates were similar in both tissues in all cases. Since the ERNI promoter is thought to be active in neuronal tissue, the recombination rate in the brain was probably expected to increase, but the recombination rate in the brain and muscle was almost always the same. Whether the observed change in Cre recombinase activity is due to a change in Cre expression rate or a change in the time at which Cre activity is expressed is unclear. For example, in a Cre4 cell line, Cre may be continuously expressed at low expression levels, resulting in a cumulative recombination rate of 100% until day 15, whereas in other cell lines it may silence Cre expression early in all cells before recombination occurs . Alternatively, Cre4 can express Cre protein very early only in early development and can catalyze 100% recombination rate.

The only cell line that did not show recombination was a cell line with a rearranged or deleted transgene.

Example  35: Different Reaper  Successful recombination of three chicken cell lines carrying inserts

To demonstrate that the Cre recombinase can catalyze the recombination of different LoxP substrates in the pLenti-ERNI-Cre transgenic chicken, the Cre4 cell line was incubated with three different LoxP-Ripper cell lines (6-03, 6-51 and 9-51 Quot;). The Cre4 cell line was chosen because it had previously had 100% recombination rate. One LoxP-ripple transplantation gene and one copy number of Cre4 transplantation genes were selected for embryo transfer.

In order to determine the recombination rate of the three LoxP-ripple transgene genes, the ability of Cre to catalyze recombination in a double transgenic embryo carrying one copy of the Cre transplant gene and one copy of the LoxP- Were analyzed by Southern blotting. The LoxP-ripper transgene contained a 1.4 kb sequence (referred to as a stop cassette) adjacent to the LoxP site in the same orientation. Recombination between the two LoxP sites cleaves the 1.4 kb sequence from the chromosome leaving a single LoxP site. Subsequently, the intervening sequence disappeared because it was no longer linked to the chromosome. After cleavage, the LoxP-ripple transgene gene was reduced in size by 1.4 kb. A Southern blot assay was developed in which size reduction of the LoxP-ripper transgene gene is used to measure Cre recombinase activity. Upon digestion with the restriction enzyme SacI, a (non-recombined) full-length LoxP-Ripper fragment of approximately 2.8 kb was produced when hybridized to a probe consisting of the ripper gene and a portion of the lentiviral vector backbone (blastididin gene and SV40 sequence) . Upon Cre-mediated recombination and cleavage of the 1.4 kb stop sequence, the Ripper SacI fragment was approximately 1.4 kb in size when hybridized with the same probe. The probe hybridized equally to both the full-length LoxP-ripple transgene and to the recombinant LoxP-lipase transgene by hybridizing to a sequence not affected by Cre recombination.

To calculate the Cre-Iox recombination rate in each LoxP-lipa cell line, the ratio of the band intensities of the (non-recombinant) full-length transgene gene to the recombinant transgene was determined. When Cre4 was able to cleave the stop cassette in all three ripper cell lines, only the recombined ripper band was observed because the LoxP-ripple transgene was recombined in all cells. The results shown in Figure 29 show that all three Ripper cell lines are 100% cleaved by the stop cassette in the presence of the Cre4 transgene.

Example  37: From the junction point integrated in the chicken genome EGFP  And Neo Cre  Intermediate cutting

Cre recombination was performed in cultured PGCs and in vitro in transgenic birds. To perform Cre-Iox recombination in cultured PGCs, cells were transiently transfected with Cre expression vectors.

DOC2 cells were used to transfect with Cre expression vectors. This PGC cell line harbored a junctional construct integrated in chromosome 21 on Prk and CpG islands linked to several ESTs. All cells in the starting DOC2 cultures fluoresced green because they retained the CX-EGFP gene in the binding site construct. Two Cre expression vectors were used: pBS 185 with the Cre gene under the transcriptional control of the human CMV promoter, or the ERNI-Cre construct with the ERNI promoter driving Cre expression.

The Cre expression construct was transiently transfected into DOC2 cells. After several days, the cultures were monitored for green fluorescence as an indicator of whether the cells had absorbed Cre constructs, expressed Cre, or whether Cre cleaved the sequence between the LoxP sites in the binding point vector, including CX-EGFP-CX-neo Respectively. After Cre transfection, cultures consisted of green cells and non-green cells. To purify the two clusters, the cultures were sorted based on green fluorescence by flow cytometry. Millions of cells were collected from each cluster (green and non-green).

Southern blot analysis was used to demonstrate that the EGFP gene was cleaved in non-green clusters of cells (see Figure 30). Genomic DNA was prepared from two cell clusters (green and non-green) and digested with HindIII restriction enzyme. The DNA was fractionated on an agarose gel, transferred to a nylon membrane and hybridized with the radiolabeled sequence from the puromycin resistant gene present at the junction. The furo gene is a region of the binding site construct that was not cleaved by Cre-Iox recombination so that the furoprobe would be able to detect fragments from genomic DNA from both DOC2-cleaving and non-cleaved cells. The HindIII fragment of the expected size is as follows: EGFP + (uncut), 5521 bp; EGFP- (cut), 1262 bp. Cleaved fragments were observed, indicating that Cre-Iox recombination in non-green cells resulted in deletion of the CX-EGFP-CX-neo sequence between the two LoxP sites in the integrated binding site construct in DOC2 cells.

Example  38: IgL pKO5B Targeting  Production of vector

To target the chicken IgL locus, a targeting vector lacking the endogenous J and C regions of the targeted integrated locus was generated (Figure 31). The vector was identical to the IgL pKO5 described above, but the selectable marker was altered. The 5 'homology region in the vector consists of a 2327 bp fragment near the IgL V region and the 3' homology region consists of a 6346 bp fragment from the downstream of the C region. Homologous cancers were cloned from homologous gene DNA obtained from cell lines used for targeted transfection. Targeting constructs contain more than one mode and destroy expression such as stop codon, nonsense sequence, attP site, or a combination thereof. In addition, the vector contained a selectable marker gene and a site-specific recombination site.

HS4 ERNI - Neo : The 804 bp neomycin resistance gene was placed under the transcriptional control of the 800 bp ERNI promoter for expression in PGC. ERNI expression in chickens should be restricted to very early embryos so that selectable markers should not be expressed in adult chickens. A 250 bp core HS4 insulator element was repeatedly duplicated from the chicken β-globin locus and redundant insulators were placed on either side of the ERNI-neo selectable marker. (For Cre-mediated recombination) a single LoxP site was cloned upstream of HS4-ERNI-neo.

LoxP = ATAACTTCGTATAGCATACATTATACGAAGTTAT (SEQ ID NO: 47)

attP - furo: 600 bp without the furo gene promoter (for phiC31 mediated recombination) was connected to the 43 bp attP site. The attP-furo was then cloned downstream of HS4-ERNI-neo.

Together, the LoxP-HS4-ERNI-neo-attP-furo screenable marker cassette was 4089 bp.

attP = ACGCCCCCAACTGAGAGAACTCAAAGGTTACCCCAGTTGGGGC (SEQ ID NO: 48)

5 ' homology arm : A 333 bp NotI-NcoI PCR product (primers 5'-NotI, TTCTTGCGGCCGCAGGGAGCCATAGCCTGCTCCCATCATGCCC (SEQ ID NO: 49) and 3'-NcoI) amplified from the 1994 bp NcoI- BamHI fragment + PGC35 genomic DNA obtained from the PGC35 IgL SacI clone NcoI, AGAGGAGCCCAGGCCATGGCGGAAT) (SEQ ID NO: 50)) to generate a 2327 bp fragment.

The PCR fragment contained the overlapping genomic Nco I site and the two fragments were ligated together.

The resulting 2327 bp fragment was released with Nod and BamHI for cloning into pKO vector backbone upstream of HS4 ERNI-furo. The NotI site was not present in the genome but was added by PCR.

3 ' homology arm : The following three fragments were ligated together to generate a 6346 kb Spel-BglII fragment: the Spel-EcoRl fragment from the SacI genomic clone plus the EcoRl-ApaLI clone from the EcoRI-Mfel genomic clone plus the EcoRI-Mfel (Primer 5 'ApaL, AGTGCAGCTGCAGTGCACGGTA (SEQ ID NO: 51) and 3'-BglII, TTCTTAGATCTGTGACAAGCAGTCTCCGGTTAACA (SEQ ID NO: 52)) amplified from the clone

The BglII site was not present in the genome but was added by PCR. The 3 'homology arm was cloned as a pKO vector backbone between HS4 ERNI-furo and HS4 [beta] -actin EGFP.

HS4 [ beta] -actin EGFP : 1.3 kb chicken β-actin promoter was used to drive the expression of the 700 bp EGFP gene. One copy of overlapping HS4 insulator was added to the ends to isolate the randomly inserted targeting vector from the location effect and express EGFP.

The final IgL pKO5B targeting vector was 17,681 bp in size and was linearized with NotI prior to transfection with PGC.

Example  39: PGC &Lt; / RTI &gt; KO -07 IgL Knockout PGC  Production of cell line

38 [mu] l of 5 x 106 cells in 100 [mu] l of electrophoresis buffer (V buffer from Amaxa) were each transfected with 10 [mu] g DNA. All droplets were electrophoresed with the Amaxa nucleofructor pulse A33 (Amaxa). Nine clones were obtained, four of which were GFP-positive and did not perform further. Five non-GFP expressing clones were extended for Southern analysis and named KO-07, KO-08, KO-09, KO-10 and KO-11.

Example  40: Southern Blot  analysis

For the 5 'side of homologous recombination, genomic DNA from 5 clone PGC cell lines transfected with IgL pKO5B was digested with SacI restriction enzyme and fractionated on 0.7% agarose gel. The DNA was transferred to a nylon membrane and hybridized with a probe (i. E., An external probe) from the chicken IgL locus upstream from the region used as the homology arm. The probe was a 0.5 kb SacI-BstEII fragment and approximately 10 kb of wild-type fragment and approximately 4 kb of mutant fragment were detected (Fig. 32, left panel). For the 3 'side of targeting, genomic DNA was solubilized by BstEII and blots were hybridized with a 3' 1.7 kb Nsil-Mfel fragment external to the targeting vector (Figure 32, right panel).

Example  41: gonads Chimeric  produce

In step 15-16 (Hamburger & Hamilton), 3000 PGCs were injected per embryo into the abdominal aorta. The embryos were cultured in surrogate shells. The hatched chicks were grown to sexual maturity.

The chimeric rooster was mated by wild-type Barred Rock hen by artificial insemination. Sperm was collected from 9 roosters and used to fertilize the hen. Six out of 30 roosters transferred the black feather phenotype to offspring and showed gonadal metastasis of IgL knockout PGC (Table 1). One of the roosters (IV75-41) metastasized at a rate of more than 50%.

Table 15. Gonadal transfer rate of IgL knockout in 9 chimera cocks with IgL knockout PGCs. (Representing gonadal transition of the knockout PGC) and the number of white followers (from host PGC) were summarized. The rooster crowed the black posterity in boldface.

Example  42: Knockout  Southern embryo Blot  analysis

On day 14, embryos of black plumage were euthanized and genomic DNA was prepared from skeletal muscle. Southern blotting was performed in which the knockout was transferred to five embryos (2, 3, 4, 6 and 7 embryos) of seven embryos tested in the experiment. Embryo No. 1 and Embryo No. 5 were wild-type embryos that inherited the wild-type IgL allele from heterozygous targeting KO-07 knockout PGC (Figure 33).

delete

delete

delete

delete

delete

delete

delete

delete

delete

delete

delete

delete

delete

                         SEQUENCE LISTING <110> Origen Therapeutics        Van de Lavoir, Marie-Cecile        Leighton, Philip   <120> Transgenic Chickens With An Inactivated Endogenous Gene Locus <130> 700603.4008 <140> PCT / US2008 / 073214 <141> 2008-08-14 <150> 60 / 964,891 <151> 2007-08-14 <150> 12 / 192,020 <151> 2007-08-14 &Lt; 150 > US 11 / 346,630 <151> 2006-02-01 <150> US 11 / 204,879 <151> 2005-08-15 &Lt; 150 > US 11 / 049,229 <151> 2005-02-01 <160> 54 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> V-1, primer to amplify a 751 bp fragment from CVH transcript <400> 1 gctcgatatg ggttttggat 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > v-2, primer to amplify a 751 bp fragment from CVH transcript <400> 2 ttctcttggg ttccattctg c 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Dazl-1, primer to amplify a 536 bp fragment from Dazl transcript <400> 3 gcttgcatgc ttttcctgct 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Dazl-2, primer to amplify a 536 bp fragment from Dazl transcript <400> 4 tgcgtcacaa agttaggca 19 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> Act-RT-1, primer to amplify a 597 bp fragment from chicken actin        transcript <400> 5 aacaccccag ccatgtatgt a 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Act-RT-2, primer to amplify a 597 bp fragment from chicken actin        transcript <400> 6 tttcattgtg ctaggtgcca 20 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Hs4-Bam-F, PCR primer <400> 7 aggatccgaa gcaggctttc ctggaagg 28 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> HS4-Bgl-R, PCR Primer <400> 8 aagatcttca gcctaaagct ttttccccgt 30 <210> 9 <211> 45 <212> DNA <213> Bacteriophage phi-C31 <220> <221> misc_feature <223> attB site, junction region <400> 9 tgcgggtgcc agggcgtgcc cttgggctcc ccgggcgcgt actcc 45 <210> 10 <211> 44 <212> DNA <213> Gallus <220> <221> misc_feature <223> cell line NLS1-41, junction region <400> 10 gcgggtgcca gggcgtgccc cgctggggcc gcactccttc atca 44 <210> 11 <211> 45 <212> DNA <213> Gallus <220> <221> misc_feature <223> cell line 5-7, junction region <400> 11 tgcgggtgcc agggcgtgcc atggtcctgt aggagtatga caggc 45 <210> 12 <211> 45 <212> DNA <213> Gallus <220> <221> misc_feature <223> cell line 19-1-1, junction region <400> 12 tgcgggtgcc agggcgtgga agccccttgg tctcagcagt gacac 45 <210> 13 <211> 45 <212> DNA <213> Gallus <220> <221> misc_feature <223> cell line 18-4-11, junction region <400> 13 tgcgggtgcc agggcgtgcc ctaaccaagg gctgaccggg acaag 45 <210> 14 <211> 45 <212> DNA <213> Gallus <220> <221> misc_feature <223> cell line 18-3-43, junction region <400> 14 tgcgggtgcc agggcgccat ggagacccca atggccccat ttaac 45 <210> 15 <211> 45 <212> DNA <213> Gallus <220> <221> misc_feature <223> cell culture 18-5-36, junction region <400> 15 tgcgggtgcc agggcgtgcc atcctatggc atcctataga accct 45 <210> 16 <211> 45 <212> DNA <213> Gallus <220> <221> misc_feature <223> cell culture 18-5-36-3, junction region <400> 16 tgcgggtgcc agggcgtgcc cactatgggg tcctacaacc actat 45 <210> 17 <211> 45 <212> DNA <213> Gallus <220> <221> misc_feature <223> cell culture NLS2-47, junction region <400> 17 tgcgggtgcc agggcgtgct ggggtcctac aaccactatg gggcc 45 <210> 18 <211> 45 <212> DNA <213> Gallus <220> <221> misc_feature <223> cell culture NLS1-30, junction region <400> 18 tgcgggtgcc agggcgtgcc ccctatgggg ccattggggt ctcca 45 <210> 19 <211> 45 <212> DNA <213> Gallus <220> <221> misc_feature <223> cell culture 18-3-12, junction region <400> 19 tgcgggtgcc agggcgtgcc cggggtccta taaccactat ggggc 45 <210> 20 <211> 45 <212> DNA <213> Gallus <220> <221> misc_feature <223> cell culture 19-5-21, junction region <400> 20 tgcgggtgcc agcatcctat ggcaccctat agaaccctat ggcat 45 <210> 21 <211> 45 <212> DNA <213> Gallus <220> <221> misc_feature <223> cell culture NLS2-38, junction region <400> 21 tgcgggtgcc agggcgtgcc cccgggcgcg tactccacct cacac 45 <210> 22 <211> 252 <212> DNA <213> Gallus <220> <221> misc_feature <223> PGC clone 18-5-36-2, insertion site <400> 22 tgcgggtgcc agggcgtgcc cactatgggg tcctacaacc actatggggc cacagaaccc 60 cctatggggc cctataacca ctatgggggc ctataacccc ccatggggtc ctataaccac 120 tatggggccc tataaccact atggggtcct ttaaccacta tggggcccca gaacccccta 180 tggggcccta tagccactat ggggtcctat aaccccccat ggggtcctgt aaccactatg 240 gggccctata ac 252 <210> 23 <211> 246 <212> DNA <213> Gallus <220> <221> misc_feature <223> PGC clone 2-47, insertion site <400> 23 tgcgggtgcc agggcgtgct ggggtggtac aaccactatg gggccacaga accccctatg 60 gggccctata accactatgg ggccctataa ccccccatgg ggtcctataa ccactatggg 120 gccctataac cactatgggg tcctttaacc actatggggc cccagaaccc cctatggggc 180 cctatatcca ctatggggtc ctataacccc ccatggggtc ctgtaaccac tatggggccc 240 tataac 246 <210> 24 <211> 268 <212> DNA <213> Galllus <220> <221> misc_feature <223> PGC clone 18-3-12, insertion site <400> 24 tgcgggtgcc agggcgtgcc cggggtccta taaccactat ggggccacag aaccccctat 60 ggggccctat aaccactatg gggccctata accccccatg gggtcctgta accactatgg 120 ggccccagaa ccccctatgg ggccctataa ccccccatgg ggtcctgtaa ccactatggg 180 gccccagaat cccctatggg gccctatagc aactatgggg tcctataacc ccccatgggg 240 tcctgtaacc actatggggc cctataac 268 <210> 25 <211> 203 <212> DNA <213> Gallus <220> <221> misc_feature <223> PGC clone 18-3-43 <400> 25 tgcgggtgcc agggcgccat ggagacccca atggccccat ttaaccccac tgaccccaat 60 gtccccaaaca tcccctgatg tccccaatgt ggccccgatg accccatgat gtccccaata 120 cccccaatga ccacaacgac cccatacccc cctgtgaccc catacccccc aatgacccca 180 tatccccgat gccccccaac gcc 203 <210> 26 <211> 260 <212> DNA <213> Gallus <220> <221> misc_feature <223> PGC clone 18-5-36-1 <400> 26 tgcgggtgcc agggcgtgcc atcctatggc atcctataga accctatggc accaaatggc 60 atcctatagc acccaatggc agccaatggc accctatggc atcctatagc atcccatggc 120 atcctatagc accccatggc atcctagagc accctatggc accctatggc atcctatggc 180 accctataga acccaatggc atcctatggc atcctatagc agcctatggc accctatagc 240 accctatggc accctatggc 260 <210> 27 <211> 263 <212> DNA <213> Gallus <220> <221> misc_feature <223> PGC clone 19-5-21 insertion site <400> 27 tgcgggtgcc agcatcctat ggcaccctat agaaccctat ggcaccaaat ggcatcctat 60 agcacccaat ggcagccaat ggtatcctat ggcatcctat ggcacccaat ggcaccctat 120 ggcatcctat ggcacccaat ggcatcctat gcccatcat ggcatcctat ggcacccaat 180 ggcatcctat ggcatcctat ggcatcctat ggcaccctat ggcatcctat ggcaccctat 240 agaaccctat gacatcctat ggc 263 <210> 28 <211> 257 <212> DNA <213> Gallus <220> <221> misc_feature <223> PGC clone 1-30 insertion site <400> 28 tgcgggtgcc agcatcctat ggcaccctat agaaccctat ggggtctctg tggggccatt 60 gggccctatg gggccgtcgg ggctctatgg ggccataggg gtctccatgg ggtctctctg 120 gggccattgg gccctatggg gccgttgggg ccctatgggc ccattagggc tctgtggggc 180 cattgggcac tatttttcct ttgggggccc tatggggcca ttggggtctc cacggggtct 240 ctctgggtcc attgggc 257 <210> 29 <211> 281 <212> DNA <213> Gallus <220> <221> misc_feature <223> PO41 consensus sequence <400> 29 tatggggctc tatggggctc tatggggctc tatggggcgg ctatggggct ctatggggct 60 ctatggggct ctatggggcg gctatggggc tctatggggc tctatggggc tctatggggc 120 ggctatgggg ctcatggggc tctatggggc tctatggggc ggctatgggg ctctatgggg 180 ctctatgggg ctctatgggg cggctatggg gctctatggg gctctatggg gctctatggg 240 gcggcgatgg ggctctatgg ggctctatgg ggctctatgg g 281 <210> 30 <211> 100 <212> DNA <213> Gallus <220> <221> misc_feature <223> attP sequence, 100 bp region of consensus repeat <400> 30 cccaggtcag aagcggtttt cgggagtagt gccccaactg gggtaacctt tgagttctct 60 aagttggggg cgtagggtcg ccgacatgac acaaggggtt 100 <210> 31 <211> 100 <212> DNA <213> Gallus <220> <221> misc_feature <223> PO41 sequence, 100 bp of consensus repeat <400> 31 ccgtatgggg ctctatgggg ctctatgggg ctctatgggg cggctatggg gctctatggg 60 gctctatggg gctctatggg gcgcctatgg ggctctatgg 100 <210> 32 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ERNI-133F, forward primer <400> 32 ttgctcaagc ccccaggaat gtca 24 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Puro-8R, reverse primer <400> 33 cgaggcgcac cgtgggcttg ta 22 <210> 34 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> ALDH3A2-3 primer <400> 34 agtggtcacc gggggagt 18 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALDH3A2-4 primer <400> 35 tcacagacac aatgggcagg 20 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Actin RT-1 primer <400> 36 aacaccccag ccatgtatgt a 21 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Actin RT-2 primer <400> 37 tttcattgtg ctaggtgcca 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> REAPER F1 primer <400> 38 caccagaaca aagtgaacga 20 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> REAPER F2 primer <400> 39 tgtttgacaa aaaattgatg c 21 <210> 40 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> ERNI-738 primer <400> 40 atgcgtcgac gtggatgttt attaggaagc 30 <210> 41 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> ERNI + 83 primer <400> 41 atgcgctagc tggcagagaa cccct 25 <210> 42 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Cre-C primer <400> 42 ccgccggaga tcttaatgcc caagaagaag aggaagctgt ccaatttact gaccgtacac 60 <210> 43 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Cre-R1 primer <400> 43 tcgaattcga atcgccatct tccagcaggc g 31 <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ERNI-37F primer <400> 44 accacggcaa cgggagaggc ttat 24 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ERNI-342F primer <400> 45 tgggcaaagg cagaggaatc 20 <210> 46 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Puro-83R primer <400> 46 gcgtggcggg gtagtcg 17 <210> 47 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> loxP marker site <400> 47 ataacttcgt atagcataca ttatacgaag ttat 34 <210> 48 <211> 43 <212> DNA <213> Bacteriophage phi-C31 <220> <221> misc_feature <223> attp site <400> 48 acgcccccaa ctgagagaac tcaaaggtta ccccagttgg ggc 43 <210> 49 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> NotI forward primer <400> 49 ttcttgcggc cgcagggagc catagcctgc tcccatcatg ccc 43 <210> 50 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Nco1 reverse primer <400> 50 agaggagccc aggccatggc ggaat 25 <210> 51 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ApaL forward primer <400> 51 agtgcagctg cagtgcacgg ta 22 <210> 52 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> BglII reverse primer <400> 52 ttcttagatc tgtgacaagc agtctccggt taaca 35 <210> 53 <211> 544 <212> DNA <213> Artificial DNA <220> <223> ALDH3A2 RT-PCR product A <400> 53 agtggtcacc gggggagtgc ctgagaccac agaactgctg gaacagaggt ttgatcacat 60 cctttacacc gggaactccg ctgtgggcaa aattgtgatg gcagcggctg ccaagcacct 120 gacacccgtc acgctggagc tgggtgggaa gagcccctgc tacatcgaca cggactgtga 180 cctggctgtc gcctgcaggc ggatagcatg ggggaagtac agaactgtgg gcaaacctgc 240 attgccccag actatgtcct gtgccacccg tccatccaga gccaggtggt ggagaacatc 300 aaggcgacgc tgcaggagtt ctatggggag gatgtgaaga agtgtccaga ttatgaaagg 360 atcatcaaca agcgtcactt caagaggatc atgaacctgc tggaagggca gaagatcgcc 420 catgggggag agactgacga ggcctcctgc ttcatagcac ccaccatcct cacggacgtt 480 tctgcggagt caaaggtgat ggaagaggag atctttgggc cagtcctgcc cattgtgtct 540 gtga 544 <210> 54 <211> 680 <212> DNA <213> Artificial DNA <220> <223> ALDH3A2RT-PCR product B <400> 54 agtggtcacc gggggagtgc ctgagaccac agaactgctg gaacagaggt ttgatcacat 60 cctttacacc gggaactccg ctgtgggcaa aattgtgatg gcagcggctg ccaagcacct 120 gacacccgtc acgctggagc tgggtgggaa gagcccctgc tacatcgaca cggactgtga 180 cctggctgtc gcctgcaggc ggatagcatg ggggaagtac atgaactgtg ggcaaacctg 240 cattgcccca gactatgtcc tgtgccaccc gtccatccag agccaggtgg tggagaacat 300 caaggcgacg ctgcaggtgg gcatggcagc tcttgtgcca tggcagttgt ccctctcacc 360 acaggggtgt cactttctgt cctggtagcc ctgatgctgc tgctaagctg aaggtgcatc 420 aaaggcttgg ttgtgctgtg tgctgtggca ggagttctat ggggaggatg tgaagaagtg 480 tccagattat gaaaggatca tcaacaagcg tcacttcaag aggatcatga acctgctgga 540 agggcagaag atcgcccatg ggggagagac tgacgaggcc tcctgcttca tagcacccac 600 catcctcacg gacgtttctg cggagtcaaa ggtgatggaa gaggagatct ttgggccagt 660 cctgcccatt gtgtctgtga 680

Claims (20)

A transgenic chicken derived from a clonally transgenic chicken primordial germ cell (PGC), wherein the genome of the PGC and the genome of the transgenic chicken are targeted constructs for destroying the J and C gene segments of an endogenous immunoglobulin light chain gene, Wherein the transgenic chicken comprises an exogenous promoter operably linked to a gene. delete delete delete delete delete 2. The transgenic chicken of claim 1, wherein the targeting construct inserts a stop codon, an att-p site, a nonsense sequence, or a combination thereof into the endogenous immunoglobulin light chain gene. 2. The transgenic chicken of claim 1, wherein the targeting construct comprises two homologous regions for an endogenous immunoglobulin light chain gene and a selectable marker located between the homologous regions. delete The transgenic chicken according to claim 1, wherein the endogenous immunoglobulin light chain gene comprises deletion of 10 kb or more. A method of producing transgenic chickens,
Introducing the targeted construct into the cultured chicken PGC to incorporate the targeted construct into the genome of the cultured primitive germ cell (PGC) to destroy the J and C regions of the endogenous immunoglobulin light chain gene to produce transformed PGCs, Wherein the targeting construct comprises a nucleotide sequence encoding a J and C segment of an immunoglobulin light chain, a nucleotide sequence encoding an exogenous promoter used to initiate expression of the transgene, and a nucleotide sequence encoding the transgene &Lt; RTI ID = 0.0 &gt; PGC &
Selecting the clonally transformed PGC,
Inserting the clonally transformed PGC into a recipient chicken embryo,
Hatching chimeric chickens from said embryos, said chickens comprising a targeting construct that destroys the J and C regions of an endogenous immunoglobulin light chain gene and a promoter derived from clonally transformed PGCs comprising a promoter operably linked to the transgene Hatching a chimeric chicken comprising germline cells, and
Breeding a chimeric chicken to produce a transgenic chicken, wherein the genome of the transgenic chicken is derived from the germline cell and comprises a targeting construct which destroys the J and C regions of the endogenous immunoglobulin light chain gene, The method comprising the steps of producing a transgenic chicken comprising an operably linked promoter
&Lt; / RTI &gt; delete delete delete delete 12. The method of claim 11, wherein the targeting construct inserts a stop codon, a nonsense sequence, an att-p site, or a combination thereof into an endogenous immunoglobulin light chain gene. 12. The method of claim 11, wherein the targeting construct comprises a selectable marker located between two or more homologous regions and two homologous regions for an endogenous immunoglobulin light chain gene. delete 12. The method of claim 11, wherein the targeting construct comprises a positive selection marker. 12. The method of claim 11, wherein the targeting construct comprises a negative selection marker.

Images