EP1334356A2 - Verfahren zur erzeugung klonierter tiere unter verwendung von chromosomen-shuffling - Google Patents

Verfahren zur erzeugung klonierter tiere unter verwendung von chromosomen-shuffling

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Publication number
EP1334356A2
EP1334356A2 EP01981389A EP01981389A EP1334356A2 EP 1334356 A2 EP1334356 A2 EP 1334356A2 EP 01981389 A EP01981389 A EP 01981389A EP 01981389 A EP01981389 A EP 01981389A EP 1334356 A2 EP1334356 A2 EP 1334356A2
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Prior art keywords
isogenic
animal
cell
haploid
chromosome
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English (en)
French (fr)
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EP1334356A4 (de
Inventor
Robert Lanza
Jose Cibelli
Philip Damiani
Michael D. West
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Astellas Institute for Regenerative Medicine
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Advanced Cell Technology Inc
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Publication of EP1334356A2 publication Critical patent/EP1334356A2/de
Publication of EP1334356A4 publication Critical patent/EP1334356A4/de
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C12N15/877—Techniques for producing new mammalian cloned embryos
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0273—Cloned vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/101—Bovine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/30—Bird
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/40—Fish
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A01K2267/025—Animal producing cells or organs for transplantation

Definitions

  • This invention concerns methods of cloning animals that incorporate methods for manipulating or shuffling chromosomes.
  • the methods find important use in the fields of agriculture, xenotransplantation, laboratory science and species conservation, where shuffling of chromosomes can be used to correct chromosomal abnormalities, and to create autosomally isogenic, sexually non-isogenic cloned animals.
  • Microcell-mediated chromosome transfer has been used for many years in order to introduce a single chromosome into a target cell.
  • Saxon et al demonstrated the complete suppression of tumorigenicity in HeLa cells by introducing a single human chromosome via microcell fusion.
  • Saxon et al. 1986, "Introduction of human chromosome 11 via microcell transfer controls tumorigenic expression of HeLa cells," EMBO J. 5: 3461-66.
  • This technology was used by others to create libraries of human chromosomes, by fusing single human chromosomes carrying the HGPRT gene with HGPRT-deficient mouse cells.
  • MACs mammalian artificial chromosomes
  • inner cell mass cells cells from the blastocyst which form both somatic and germ cells
  • inner cell mass cells were found to support a low rate of development to the blastocyst stage with some offspring obtained.
  • trophectodermal cells the cells from the blastocyst that form the placenta
  • trophectodermal cells did not support the development of the nuclear fusion to the blastocyst stage.
  • somatic cells to be used for nuclear transfer could be used to facilitate complex genetic manipulations of donor cells, and particularly the replacement of chromosomes in cloned animals.
  • the present invention makes use of somatic cell donor cells for nuclear transfer to create complex chromosomal arrangements, and particularly chromosomal replacements, in cloned and transgenic animals.
  • This technology may be used to produce cloned cells, embryos, blastocysts, fetuses and animals that are autosomally isogenic and sexually non-isogenic, in order to make a population more uniform or improve quality control in xenotransplantation.
  • Such chromosome shuffling techniques can also be used to eliminate chromosomal abnormalities, such as inversions or translocations from the clone of an animal, produce a sexual mate for an extinct animal where the genome of only one animal is extant, or to produce the opposite sex of an existing animal or embryo where the genome of only one sex is available or desired.
  • somatic cells from the desired animal are isolated, and one X chromosome is removed and replaced with a Y chromosome from another animal.
  • both X chromosomes may be replaced with the sex chromosomes - one X and one Y - from another mammal.
  • Multiple females may be cloned from the original somatic cells, and males would be produced from the autosomally isogenic sexually non- isogenic (AISN) cells.
  • AISN autosomally isogenic sexually non- isogenic
  • the cloned bulls could then be used to breed females by sexual reproduction rather than by cloning.
  • semen from these males can be frozen for use in artificial insemination in order to produce more females having the desired trait.
  • the male AISN animals may be genetically modified to produce only female animals using the technology described in Application Serial No. 60/184,830, now PCT/US01/05932, herein incorporated by reference in its entirety. This would allow the marketing of semen and the ready propagation of female animals having a desirable genetic make-up while simultaneously preventing customers from breeding their the AISN animals on their own without exchange of compensation for the technology.
  • single sex technology can also be used to produce all male offspring, and female AISN animals could be marketed as a business strategy.
  • the chromosome shuffling techniques of the present invention may also be combined with nuclear transfer techniques designed to create homozygous diploids of desirable haploid genomes, in order to achieve allelically isogenic breeding pairs of animals that differ only as to their sex chromosomes, i.e., each is a complete autosomal homozygous diploid.
  • allelically isogenic animals results in isogenic male and female offspring without the need for years of inbreeding or successive cloning in order to generate animals. Further, such breeding avoids the potential genetic diversity associated with sexual reproduction between cloned breeding pairs where crossing over and chromosomal segregation can result in the appearance of undesirable recessive traits in the progeny.
  • allelically isogenic breeding pairs will have significant utility in the agricultural field where it is often desirable to propagate animals with specific traits such as high milk output, milk with specific lipid or protein profiles, or animals which produce meat, leather, wool or fiber having a desired characteristic.
  • breeding pairs would also find utility in laboratory settings as well as xenotransplantation studies, where lowering the statistical "noise" from genetic diversity, or eliminating the risk of introducing viral contaminants is desirable.
  • Autosomally and allelically isogenic breeding pairs provide the ultimate business model, whereby purchasers and handlers can be assured that desirable animals may be easily maintained via sexual reproduction or artificial insemination without the need for nuclear transfer techniques.
  • Methods of making autosomally isogenic, allelically isogenic diploid nuclear transfer units are also encompassed, as are methods of making cells, oocytes, balstocysts, inner cell masses, ES cells, embryos, and individual animals having the same characteristics.
  • An object of the invention is also to provide business methods for using the breeding pairs to mass produce animals that have been genetically modified, bred or selected to provide an advantage in a desired market, as well as business methods to maintain control over the breeding of such animals by marketing animals and/or semen that can only be used by purchasers and handlers to produce animals of a single sex.
  • FIG. 1 In protocol for Percoll separation of somatic cells from semen, diagram depicting Percoll layers prior to (A) and following (B) centrifugation.
  • the present invention concerns the use of chromosomal replacement techniques in the context of producing cloned and transgenic animals, in order to correct chromosome abnormalities or alter autosomal genotypes, and provide for novel breeding pairs by replacing the sex chromosome in animals to be cloned.
  • Replacement of a sex chromosome, or an X or Y chromosome will result in animals that are autosomally isogenic and sexually non-isogenic (AISN), with "autosomally isogenic” meaning that the paired sets of autosomes (non-sex chromosomes) in each animal are isogenic or identical.
  • animals that are both "autosomally” and “allelically” isogenic whereby each particular pair of chromosomes is internally isogenic or identical within a single animal as well as between animals.
  • the invention therefore encompasses methods of altering the sex of a cloned animal, or an animal to be cloned, or an embryo, blastocyst, fetus or cell comprising:
  • a sex chromosome When a sex chromosome is removed according to the present invention, it may be either an X or a Y chromosome, and it may be replaced by the alternative sex chromosome from a non-isogenic allogeneic animal, or even a non-isogenic, xenogeneic animal.
  • the Y chromosome In the case where the somatic cell of interest is from a male animal, the Y chromosome may be replaced by the X chromosome from another copy of the somatic cell to yield a cell with two X chromosomes.
  • the somatic cell may need to be isolated from a sample of frozen cells.
  • somatic cells preferably semen cells, may be frozen in preparation for the methodology of the invention.
  • the alternative chromosome may be taken from a xenogeneic animal, preferably one that is closely related to the extinct animal.
  • Application Serial No. pertains specifically to the cloning of endangered species, which material is hereby incorporated in its entirety.
  • the chromosome to be replaced may be removed by any feasible technique.
  • the unwanted chromosome may be removed by targeting by homologous recombination a gene or DNA sequence that results in loss of the chromosome upon mitosis or meiosis.
  • chromosomal instability results when sequences are introduced which function as a centromere. Such sequences cause a dicentric chromosome to be created, which undergoes breakage potentially leading to loss of the chromosome during cell division. Loss of chromosomes that have been genetically modified with additional centromeric sequences can be detected by karyotype analysis.
  • Cells which lose the targeted chromosome may be also be selected by including a negative selectable marker such as thymidine kinase whereby cells retaining the chromosome or pieces of the chromosome will not survive under selective conditions (i.e., gancyclovir in the case of thymidine kinase).
  • a negative selectable marker such as thymidine kinase whereby cells retaining the chromosome or pieces of the chromosome will not survive under selective conditions (i.e., gancyclovir in the case of thymidine kinase).
  • somatic cells as nuclear donors are that they may be expanded readily in culture prior to chromosome shuffling techniques.
  • embryonic cells may also be used, as may the nuclei of somatic cells, which are advantageous in that they may be preserved in a preservative (such as alcohol) prior to nuclear transfer, i.e., stored for future use.
  • Preferred somatic cells will be proliferating, i.e., in a proliferative state, but need not necessarily be expanded in culture.
  • the somatic cells may be genetically altered in other ways prior to or subsequent to chromosome exchange.
  • said cells may be modified with a chromosomal insertion or deletion, where a transgenic animal is desired that produces specific proteins in its bodily fluids or mammary glands, or where it is desirable to remove or mutate genes involved in xenotransplantation rejection.
  • the alternative sex chromosome to be introduced may also be genetically altered from its native state.
  • the methods of the present invention may be performed with a wide variety of animals, including mammals, fish, reptiles or birds.
  • Preferred animals for agricultural and xenotransplantation uses to be made by the present invention are ungulates selected from the group consisting of bovine, porcine, sheep and goat.
  • Preferred extinct or endangered animals to be reconstituted by the methods of the present invention include the gaur, bucardo, giant panda, cheetah, African bongo antelope, Sumarran tiger, Giant panda, Indian desert cat, moufion sheep and rare red deer.
  • Preferred animals to be generated for laboratory use include mouse, hamster, guinea pig and primates. The methods may also be used to clone cats, dogs, horses or other companion animal, or breed champion lines of such mammals.
  • the chromosomes to be inserted according to the claimed methods may be inserted via microcell-mediated chromsome transfer, or any other suitable technique known in the art, e.g., via injection.
  • Methods for the preparation and fusion of microcells containing single chromosomes are well known. See, e.g., U.S. Patent Nos 5,240,840; 4,806,476; 5,298,429 (herein incorporated by reference in their entirety; see also Fournier, 1981 , Proc. Natl. Acad. Sci. USA 78: 6349-53; Lambert et al., 1991, Proc. Natl. Acad. Sci. USA 88: 5907-59; Yoshida et al., 1994, J.
  • Chromosomes to be introduced into cloned cells or cells to be cloned will preferably include a selectable marker, such as aminoglycoside phosphotransferase, for example, so that cells receiving the chromosome via microcell fusion may be readily selected from those that do not.
  • a selectable marker such as aminoglycoside phosphotransferase, for example.
  • Siden and colleagues describe the construction of a panel of four microcell hybrids containing four separate insertions of the exogenous neomycin resistance gene into mouse chromosome 17. See Siden et al., 1989, Somat. Cell Mol. Genet. 15(3): 245-53.
  • U.S. Patent No. 6,133,503 also describes methodology for producing microcells by treating a host donor cell with a mitotic spindle inhibitor such as colchicine, which results in the formation of micronuclei, then with cytochalasin B, which results in the extrusion of microcells which contain one or a few chromosomes.
  • a mitotic spindle inhibitor such as colchicine
  • cytochalasin B which results in the extrusion of microcells which contain one or a few chromosomes.
  • the methods of U.S. Patent No. 5,635,376 are also helpful in the context of the present invention, in that this patent provides for female muntjac cell lines in which there is, for example, a ten-fold difference in chromosomal size between the diploid muntjac chromosomes and human chromosome 11.
  • these female muntjac cell lines are useful for the amplification of desired chromosomes prior to use in cells to be cloned because desired chromosomes may be purified to apparent homogeneity from the resulting hybrids using conventional equipment given the large size difference between the chromosome of interest and the muntjac chromosomes.
  • These patents are herein incorporated by reference in their entirety.
  • the cloned animals, embryos, blastocysts, fetuses and cells produced by the methods described herein are also part of the invention, as are the sexual mates and breeding pairs produced and their offspring.
  • the present invention includes methods of making an autosomally isogenic, allelically isogenic breeding pair of animals comprising:
  • Such methods may be further supplemented by ensuring that the breeding pair of animals only produces animals of a single sex, by also including a step or steps whereby a nucleic acid construct is introduced into at least one sex chromosome of the germ line of said male animal, wherein said nucleic acid construct encodes a transgene which is expressed post-meiotically in developing spermatids, and wherein expression of said transgene alters the fertility of sperm resulting from said developing spermatids, such that said male produces progeny of a single sex.
  • a nucleic acid construct is introduced into at least one sex chromosome of the germ line of said male animal, wherein said nucleic acid construct encodes a transgene which is expressed post-meiotically in developing spermatids, and wherein expression of said transgene alters the fertility of sperm resulting from said developing spermatids, such that said male produces progeny of a single sex.
  • Inducing meiosis to produce a haploid cell from a somatic cell may be accomplished by any successful method.
  • meiosis is accomplished by nuclear transfer of said somatic cell or the nucleus from said somatic cell (2n) into a metaphase II enucleated oocyte, and activating said nuclear transfer unit to extrude a polar body (n), thereby resulting in a haploid activated nuclear transfer unit.
  • Activation may be accomplished by exposing said nuclear transfer unit to one or more treatments selected from the group consisting of hyaluronidase, ethanol, cytochalasin B, Ca 2+ ions, change in osmolarity, electrical pulse, bohemine, ionomycin and sperm factor.
  • treatments selected from the group consisting of hyaluronidase, ethanol, cytochalasin B, Ca 2+ ions, change in osmolarity, electrical pulse, bohemine, ionomycin and sperm factor.
  • Diploid cells containing isogenic alleles may be made by allowing the activated haploid oocyte to develop to at least the two cell stage, isolating and/or separating the cells, and fusing two allelically isogenic haploid cells from said developing activated oocyte into an enucleated metaphase II oocyte.
  • the homozygous diploid may be made by isolating one haploid cell and allowing it to advance to the G2 phase of the cell cycle, at which point it is 2n or transiently diploid, and may be used as the donor nucleus for nuclear transfer.
  • Some researchers have used cytochalasin B to induce diploidization of a female pronucleus following removal of the male pronucleus from a fertilized egg.
  • allelically isogenic AISN breeding pairs include several embodiments. For instance, included are methods of making an autosomally isogenic, allelically isogenic breeding pair of animals comprising:
  • Also included are methods of making an autosomally isogenic, allelically isogenic breeding pair of animals comprising:
  • Also included are methods of making an autosomally isogenic, allelically isogenic breeding pair of animals comprising:
  • a Y chromosome isolated from a male animal inserting a Y chromosome isolated from a male animal; (7) using nuclear transfer to create a first male animal that is autosomally isogenic, allelically isogenic and sexually non-isogenic to said haploid cell by fusing an isolated haploid cell or the nucleus therefrom selected from the expanded haploid cells of step (3) and the haploid cell or the nucleus therefrom from the haploid cell of step (7) with an enucleated metaphase II oocyte;
  • Also included are methods of making an autosomally isogenic, allelically isogenic breeding pair of animals comprising:
  • the female animal is made by fusing two isolated haploid cells or the nuclei therefrom containing X chromosomes selected from the expanded haploid cells of step
  • step (7) (4) or the expanded haploid cells of step (7) with an enucleated metaphase II oocyte in order to create one animal that has two X chromosomes OR by fusing one isolated haploid cell at the G2 cell cycle stage containing an X chromosome with an enucleated metaphase II oocyte in order to create one animal that has two X chromosomes;
  • the male animal is made by fusing one isolated haploid cell having an X chromosome with one isolated haploid cell having a Y chromosome with an enucleated metaphase II oocyte in order to create one animal that has an X and a Y chromosome; wherein sexual reproduction between said first animal and said second animal produces offspring that are autosomally isogenic and allelically isogenic to said first and second animal.
  • the nuclear transfer units made by the methods of the present invention are also included.
  • a female allelically isogenic diploid nuclear transfer unit may be made by a method comprising:
  • step (3) one haploid cell from step (3) at the G2 cell cycle stage; with an enucleated metaphase II oocyte in order to create a female allelically isogenic diploid nuclear transfer unit.
  • the method is performed such that the diploid nuclear transfer unit created at step (4) is activated such that there is no extrusion of a polar body.
  • Activated diploid nuclear transfer units may further develop into an allelically isogenic cells, blastocysts, inner cell masses, ES cells, embryos, fetuses or animals.
  • Methods of making male autosomally isogenic, allelically isogenic diploid nuclear transfer units are also included, and such methods may be performed by: (1) isolating a somatic cell from a preferred animal; (2) inducing meiosis of said somatic cell by nuclear transfer of said somatic cell or the nucleus from said somatic cell (2n) into a metaphase II enucleated oocyte and activating said nuclear transfer unit to extrude a polar body (n), thereby resulting in a haploid activated nuclear transfer unit; (3) allowing said haploid activated nuclear transfer unit to differentiate and expand to at least the two cell stage;
  • the cell made in step (4) with an enucleated metaphase II oocyte in order to create a male autosomally isogenic, allelically isogenic diploid nuclear transfer unit.
  • the allelically isogenic diploid nuclear transfer unit made by the methods of the invention are also encompassed. Because the methods described herein enable one to pass the advantages of the cloning technology to the agricultural and other industries while at the same time enable the control over the dissemination of genetically engineered molecules to remain with the inventor or the assignee, the methods described herein are particularly useful business models. Accordingly, the invention also includes business methods for producing uniform, isogenic animals, comprising:
  • Female animals and/or said male animals may be genetically modified, bred or selected to provide an advantage in a desired market. For instance, in the agricultural market, female animals may be genetically modified, bred or selected to produce a high milk output, milk with specified lipid or protein profile, milk that contains a therapeutic protein, or milk with superior nutritional value. Alternatively, female and/or male animals may be genetically modified, bred or selected to produce meat, leather, wool or fiber having a desired characteristic.
  • target markets include laboratories, where there is a need for isogenic animals including rats, monkeys, rabbits, mice, guinea pigs to remove the statistical noise from experimentation and trials for the development of therapeutic drugs.
  • a target market would also include a xenotransplantation facility, where animals such as cows, pigs and primates are developed to provide compatible organs for human transplantation.
  • female animals and/or male animals may be genetically modified with a specific human HLA type profile, or modified such that native proteins that cause graft rejection are deleted, modified or replaced with proteins that do not cause graft rejection in humans.
  • male animal has been genetically modified such that it only produces offspring of a single sex, i.e., such that it only produces female offspring.
  • Such a model is useful where only female uniform, isogenic animals are sold commercially. Frozen semen from a male isogenic animal may also be isolated and sold to purchasers of female uniform, isogenic animals such that artificial insemination may be used to create further uniform, isogenic animals.
  • Male animals according to the invention may also be genetically modified such that they only produce male offspring, or such that they produce no offspring. This would be useful where only male uniform, isogenic animals are sold commercially. A single female isogenic animal could then sold or leased by purchasers of male uniform, isogenic animals such that purchasers may breed said female with a male in order to create further male uniform, isogenic animals.
  • the uniform, isogenic animals produced in the business methods described herein are also included in the invention, as is semen, and kits containing frozen semen for artificial insemination.
  • somatic cells can then be propagated, cryopreserved, or used as somatic cell donors for the production of nuclear transfer embryos and calves.
  • An alternative approach would be to use a Fluorescence Cell Sorter machine, which can separate sperm from somatic cells based upon DNA content.
  • fetuses may be extracted at 40 days, and fetal fibroblasts isolated and frozen. From these fetal fibroblasts, the final animals can be cloned.
  • Cells can be isolated in a similar manner from other fluids such as milk, blood or urine where such samples have been saved.
  • such cells can be cultured from frozen tissue such as skin biopsy, skeletal muscle, or whole frozen animals.
  • Step 1 In a sterile 15 ml conical centrifuge tube, layer 2 ml 90% Percoll then carefully layer 2 ml of 45% Percoll on top of the 2 ml of 90% Percoll layer as shown in the diagram below. It is best to use either a 1000 ul pipette or a 9 ml pastuer pippete. It is very critical to have a very defined interface between the two layers. This will be observed clearly because the 45% Percoll is pinkish in hue and the 90% Percoll is clear. A very defined interface will be observed if layered correctly (see Figure 1A). Step 2: Thaw semen in 35°C water for 1 min. Record all information from semen straw, including bull name and registration numbers and collection date into your laboratory notebook.
  • Step 3 Thoroughly dry the straw of semen with a KemWipe wet with ethanol and then snip end of semen straw with a clean scissor. Place the open end into a clean 15 ml conical tube. Then carefully snip off the plug end of the straw and deposit all semen into tube.
  • Step 4 With a 500 ul pipette, carefully layer all of the semen onto the top of the Percoll layers.
  • Step 5 Centrifuge at 700 x g (2000 rpm using a 6.37 inch tip radius) for 30 minutes.
  • Step 6 After centrifugation, a sperm pellet will be observed at the bottom of the 90% Percoll layer as shown in .diagram below ( Figure IB).
  • Step 7 Aspirate off the Percoll gradients leaving the sperm pellet in the tip of the tube. This is usually about only 200 ul of pellet (this will vary depending on the number of semen straws thawed).
  • Step 8 With a clean pipette tip, move the pellet into either a 35 mm tissue culture treated plate or a 4 well Nunc plate with complete DMEM medium.
  • Step 9 Remove the medium the following day and add fresh medium to the plates.
  • Step 10 Carefully observe the plates for the presence of cells - this will depend on the semen, usually 7- 14 days after the initial plating.
  • Step 11 follow standard Cell Culture Techniques once a cell line is observed.
  • Modifiedsperm TL (lOx stock used to prepare 90% Percoll) A. Ingredients 1. 3.09 ml IM KCl.

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EP01981389A 2000-10-06 2001-10-05 Verfahren zur erzeugung klonierter tiere unter verwendung von chromosomen-shuffling Withdrawn EP1334356A4 (de)

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DE10248361A1 (de) * 2002-06-30 2004-01-22 Beisswanger, Roland, Dr. Unterdrückung eines Geschlechtes durch die Veränderung von Geschlechtschromosomen
CN114457360B (zh) * 2022-02-11 2023-08-25 中国华能集团清洁能源技术研究院有限公司 一种无隔膜微电解槽放大设备及加工方法和应用

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WO2000042174A1 (en) * 1999-01-13 2000-07-20 Ppl Therapeutics (Scotland) Limited Double nuclear transfer method and results thereof
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EP1334356A4 (de) 2004-10-06
WO2002029002A3 (en) 2002-09-19
CA2425100A1 (en) 2002-04-11
AU2002213033A1 (en) 2002-04-15
WO2002029002A2 (en) 2002-04-11

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