Method for generating cloned animals using chromosome shuffling

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. Also included in the invention are 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. Such animals are particularly useful in generating a line of cloned mammals using sexual reproduction, without having to undergo nuclear transfer in order to propagate cloned animals.

DETAILED DESCRIPTION OF THE INVENTION The present invention corcerns 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. Also included in the invention are 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: (1) isolating a somatic cell from an animal to be cloned; (2) removing or programming for removal one sex chromosome from said somatic cell; (3) inserting the alternative sex chromosome from a non-isogenic animal; and (4) using nuclear transfer to create an autosomally isogenic, sexually non-isogenic animal, embryo, blastocyst, fetus or cell. Such methods may also be used in instances whereby an offspring of a particular sex is desired as a result of sexual reproduction, where the method includes: (1) isolating a fertilized ovum, embryo or blastocyst; (2) testing the sex of said ovum, embryo or blastocyst; (3) removing or programming for removal the sex chromosome from one cell of said ovum, embryo or blastocyst if it is not of the desired sex; (4) inserting the alternative sex chromosome isolated from an allogeneic animal; (5) using nuclear transfer to create an autosomally isogenic, sexually non-isogenic embryo-or blastocyst; and (6) implanting said embryo or blastocyst into a surrogate female to isolate an animal having a desired sex. 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. 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. Also encompassed are methods of producing a sexual mate for an extinct or endangered animal by removing or programming for removal one sex chromosome from said somatic cell and inserting the alternative sex chromosome from a non-isogenic animal, and using nuclear transfer to create an autosomally isogenic, sexually non-isogenic animal mate for an extinct or endangered animal. In this embodiment, particularly for extinct animals, the somatic cell may need to be isolated from a sample of frozen cells. In cases where an animal is endangered or nearing endangered levels, somatic cells, preferably semen cells, may be frozen in preparation for the methodology of the invention. Where the animal is extinct and frozen cells for replacement chromosomes do not exist, the alternative chromosome may be taken from a xenogeneic animal, preferably one that is closely related to the extinct animal. In this regard, PCT/US01/31218 filed on Oct. 5, 2001 pertains specifically to the cloning of endangered species, which material is hereby incorporated in its entirety. Also encompassed are methods of eliminating chromosomal abnormalities from the clone of an animal a damaged chromosome from a somatic cell is removed or programmed for removal, and a non-damaged chromosome from a non-isogenic animal is inserted. Nuclear transfer is then used to create an animal, embryo, blastocyst, fetus or cell from said chromosomally corrected somatic cell. The chromosome to be replaced may be removed by any feasible technique. For instance, 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. As discussed in U.S. Pat. Nos. 5,270,201 and 6,077,697, 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). As noted above, an advantage of using somatic cells as nuclear donors is that they may be expanded readily in culture prior to chromosome shuffling techniques. However, 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. For instance, 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, mouflon 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 chromosome 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. Pat. 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. NatI. Acad. Sci. USA 88: 5907-59; Yoshida et al., 1994, J. Surg. Oncol. 55:170-74; Dong et al., 1996, World J. Urol. 14: 182-89. 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. In this regard, 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. Pat. 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. The methods of U.S. Pat. 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. Thus, 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. Also included are the individual replacement chromosomes used for the present invention and any DNAs used to make genetic modifications, as well as any intermediary cell lines such as muntjac cell lines used to amplify the desired replacement chromosomes. As described briefly above, in certain embodiments, particularly business models where isogenic animals are to be produced via sexual reproduction or artificial insemination, it is desirable that the animals be allelically isogenic as well as autosomally isogenic. Accordingly, the present invention includes methods of making an autosomally isogenic, allelically isogenic breeding pair of animals comprising: (1) isolating a somatic cell from a preferred animal; (2) inducing meiosis to produce a haploid cell from said somatic cell; (3) making a diploid cell from said haploid cell which contains isogenic alleles; (4) expanding said diploid cell; (5) isolating a copy of said diploid cell or the nucleus therefrom; (6) removing one sex chromosome from said copy of said isolated diploid cell; (7) inserting the alternative sex chromosome from a non-isogenic animal; (8) using nuclear transfer to create a first animal that is autosomally isogenic, allelically isogenic and sexually non-isogenic to said allelically isogenic diploid cell; and (9) using nuclear transfer to create a second animal that is autosomally isogenic, allelically isogenic and sexually isogenic to said allelically isogenic diploid cell, 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. 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. Such methods are described in copending application Ser. No. 60/184,830, which is herein incorporated by reference in its entirety. Inducing meiosis to produce a haploid cell from a somatic cell may be accomplished by any successful method. Preferably, meiosis is accomplished by nuclear transfer of said somatic cell or the nucleus from said somatic cell (2n) into a metaphase 11 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&plus; ions, change in osmolarity, electrical pulse, bohemian, ionomycin and sperm factor. The fact that haploid oocytes, when activated, form morphologically normal blastocysts has been documented by several researchers. See Kaufman, 1982, J. Embryol. Exp. Morphol. 71: 139-54 (reporting activation with 7% ethanol); Mann and Lovell-Badge, 1984, Nature 310(5972): 66-7; O'Neill and Kaufman, 1988, 248(1): 125-31 (reporting activation with hyaluronidase); De Sutter et al., 1992, J. Assist. Reprod. Genet. 9(4): 328-37 (activation using puromycin); Henery and Kaufman, 1992, Mol. Reprod. Dev. 31(4): 258-63 (activation in 7% ethanol); Kim et al., 1997, Zygote 5(4): 365-70 (activation by ethanol plus cytochalasin B); Escriba and Garcia-Ximenez, 1999, Theriogenology 51(5): 963-73, and 2000, Anim. Reprod. Sci. 28: 59(1-2): 99-107 (activation by altering the osmolarity and Ca 2&plus; concentration with electrical pulses in mannitol medium); and Alberio et al., 2000, Mol. Reprod. Dev. 55(4): 422-32 (reporting that bohemian with or without ionomycin produces activated haploid oocytes). 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 11 oocyte. Alternatively, 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. See Markert and Petters, 1977, “Homozygous mouse embryos produced by microsurgery,” J. Exp. Zool. 201(2): 295-302; see also Anderegg and Markert, 1986, “Successful rescue of microsurgically produced homozygous uniparental mouse embryos via production of aggregation chimeras,” Proc. Natl. Acad. Sci. USA 83(17): 6509-13. Kono and colleagues have also shown that heterozygous bispermic androgenones (eggs with two Y-chromosomes made by fertilizing enucleated oocytes in vitro), also develop to the blastocyst stage. See Kono et al., 1993, Mol. Reprod. Dev. 34(1): 43-6. Thus, specific methods of making allelically isogenic AISN breeding pairs according to the present invention include several embodiments. For instance, included are methods of making an autosomally isogenic, allelically isogenic breeding pair of animals comprising: (1) isolating a somatic cell from a preferred female animal; (2) inducing meiosis to produce a haploid cell from said somatic cell; (3) expanding said haploid cell; (4) isolating a copy of said haploid cell or the nucleus therefrom; (5) removing the X chromosome from said copy of said isolated haploid cell; (6) 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 11 oocyte; (8) using nuclear transfer to create a second animal that is autosomally isogenic, allelically isogenic and sexually isogenic to said haploid cell, by fusing two isolated haploid cells or the nuclei therefrom selected from the expanded haploid cells of step (3) with an enucleated metaphase 11 oocyte; 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. Also included are methods of making an autosomally isogenic, allelically isogenic breeding pair of animals comprising: (1) isolating a somatic cell from a preferred male animal; (2) inducing meiosis to produce a haploid cell from said somatic cell; (3) selecting a single haploid cell and determining whether it contains an X or Y chromosome; (4) expanding said haploid cell; (5) isolating a copy of said haploid cell or the nucleus therefrom; (6) removing the sex chromosome from said copy of said isolated haploid cell; (7) inserting the alternative sex chromosome into said copy of said haploid cell wherein the alternative sex chromosome is isolated from either a non-isogenic animal or the original preferred animal or another haploid cell produced from said somatic cell and optionally expanding said haploid copy if an X chromosome is inserted; (8) using nuclear transfer to create two animals that are autosomally isogenic, allelically isogenic and sexually non-isogenic by fusing isolated haploid cells or the nuclei therefrom from the expanded haploid cells of step (4) and/or the haploid cell or cells or the nuclei therefrom from the haploid cell or cells of step (7) with an enucleated metaphase II oocyte in order to create one animal that has two X chromosomes and 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. Also included are methods of making an autosomally isogenic, allelically isogenic breeding pair of animals comprising: (1) isolating a somatic cell from a preferred female animal; (2) inducing meiosis to produce a haploid cell from said somatic cell; (3) expanding said haploid cell; (4) isolating a copy of said haploid cell or the nucleus therefrom; (5) removing the X chromosome from said copy of said isolated haploid cell; (6) 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 11 oocyte; (8) using nuclear transfer to create a second animal that is autosomally isogenic, allelically isogenic and sexually isogenic to said haploid cell, by fusing an isolated cell in the G2 cell cycle phase (2n) or the nucleus therefrom selected from the expanded haploid cells of step (3) with an enucleated metaphase 11 oocyte; 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. Also included are methods of making an autosomally isogenic, allelically isogenic breeding pair of animals comprising: (1) isolating a somatic cell from a preferred male animal; (2) inducing meiosis to produce a haploid cell from said somatic cell; (3) selecting a single haploid cell and determining whether it contains an X or Y chromosome; (4) expanding said haploid cell; (5) isolating a copy of said haploid cell or the nucleus therefrom; (6) removing or the sex chromosome from said copy of said isolated haploid cell; (7) inserting the alternative sex chromosome isolated from a non-isogenic animal or the original preferred animal or another haploid cell produced from said somatic cell and optionally expanding said haploid copy if an X chromosome is inserted; (8) using nuclear transfer to create two animals that are autosomally isogenic, allelically isogenic and sexually non-isogenic, wherein (a) 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 (4) or the expanded haploid cells of step (7) with an enucleated metaphase 11 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 11 oocyte in order to create one animal that has two X chromosomes; and (b) 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 11 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. For instance, a female allelically isogenic diploid nuclear transfer unit may be made by a method comprising: (1) isolating a somatic cell from a preferred female 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; and (4) fusing either (a) two haploid cells from step (3); or (b) one haploid cell from step (3) at the G2 cell cycle stage; with an enucleated metaphase 11 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 11 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; (4) replacing the sex chromosome in one cell from taken from said differentiated and expanded haploid cells using microcell-mediated chromosome transfer from the sex chromosome from a non-isogenic animal or from another haploid or somatic cell from said preferred animal if said preferred animal was a male; (5) fusing two haploid cells: (a) one from the expanded cells of step (3); and (b) the cell made in step (4); with an enucleated metaphase 11 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: (1) producing autosomally isogenic and allelically isogenic male and female animals according to the methods described herein; and (2) breeding said male and female animals to produce uniform, isogenic animals. 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. Other 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. For instance, 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. One of the most effective business models is where the 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. The skilled artisan will envision variations to the methods disclosed herein without departing from the scope of the invention. 
 Example 1 Isolation of Somatic Cells from Semen The cloning of animals by nuclear transfer has many applications in such diverse fields as agriculture, medicine and the preservation of endangered species. One difficulty commonly faced, however, is an adequate source of somatic cells. In the case of agricultural species such as cattle, highly-valued studs are often lost with no known preservation of the genome for cloning. This invention describes a technique to isolate viable somatic cells from semen, urine, milk and other sources where the isolation of somatic cells is problematic. While semen is often thought of as being largely a solution of spermatozoa that are haploid, somatic diploid cells may occasionally be shed as well. We centrifuged 0.75 ml of bovine semen at 700× g (45%-90% percoll gradient for 30 minutes), aspirated the supernatant, and resuspended the pellet of 500 ml in DMEM medium with 15 FCS. The resulting cell suspension was then plated in 35 mm 2 tissue culture plate. The culture dishes were aspirated, washed and refed 24 hours (after and every other day following). After five days of culture, fibroblastic cells were observed attached to the tissue culture dish. These 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. To reduce the chance of spontaneous abortion, 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. In addition, such cells can be cultured from frozen tissue such as skin biopsy, skeletal muscle, or whole frozen animals. The success of this method can be explained perhaps by analyzing the method of semen processing for the purpose of freezing and later use in artificial insemination. During extraction, an artificial vagina is used to collect the ejaculate and perhaps some of the cells that are around the penis along with free somatic cells originating in the accessory glands, ducts and testicle themselves will be mingled with the ejaculate. This technique will allow bulls to be “resurrected” in instances where the bulls are no longer alive but their frozen semen is available. The method is reproduced in detail below: A. Establishment of Cell Lines from Cryopreserved Semen NOTE: Please wear gloves for every step of the procedure to prevent cross contamination of samples. Percoll separation of sperm (performed at room temperature) 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. 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 scissors. 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 × 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. 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. Stock Solutions 45% Percoll Solution A. Ingredients 1. 1.5 ml 90% Percoll Stock Solution. 2. 1.5 ml Sperm TL with BSA. B. Procedure 1. Use aseptic techniques. 2. Transfer ingredients to a sterile tube. 3. Invert to mix. 4. Do not attempt to filter. Sperm TL Without BSA A. Ingredients 1. 25 ml sperm TL stock. 2. Adjust pH to 7.4 with 1 M HCl. 3. Filter sterilize 4. Prepare daily. Modified Sperm TL (1 Ox stock used to prepare 90% Percoll) A. Ingredients 1. 3.09 ml 1 M KCI. 2. 2.92 ml 0. IM NaH 2 PO 4 3. 4.675 gm NaCl 4. 2.380 gm Hepes B. Procedure 1. Add prescribed amounts of KCI and NaH 2 PO 4 solutions to ˜50 ml H 2 0 in volumetric flask. 2. Add NaCl and Hepes. 3. Adjust water to 100 ml. 4. Adjust pH to 7.3. 5. Filter sterilize and transfer to a glass bottle. 6. Store refrigerated indefinitely. 7. Readjust pH as needed. 1 M CaCl 2 -used in making 90% Percoll A. Ingredients 1. 735 mg CaCl2*2H 2 O. 2. Reagent grade water. B. Preparation 1. Weigh CaCl 2 . 2. Add 5 ml H 2 O. 3. Filter sterilize or autoclave. 4. Store in glass bottle indefinitely. 0.1 M MgCl 2 -used in making 90% Percoll A. Ingredients 1. 20.3 mg MgCl 2 *6H 2 O. 2. Reagent grade water. B. Preparation 1. Weigh MgCl 2 . 2. Add10 ml water. 3. Filter sterilize or autoclave. 4. Store in glass bottle indefinitely. 90% Percoll Solution A. Ingredients 1. 45.0 ml Percoll 2. 5.0 ml Modified Sperm TI (1 Ox stock) 3. 0.0985 ml 1 M CaCl 2 4. 0.197 ml 0.1M MgCl 2 5. 0.184 ml Lactic Acid (60% syrup) 6. 104.5 mg NaHCO 3 B. Procedure 1. Combine ingredients while stirring. 2. Store refrigerated. 3. Do not attempt to filter. 1. Sperm TL Stock 1 Final Compound mM mg/100 ml mg/500 ml NaCl 100 582 2910 KCl 3.1 23 115 NaHCO 3 25 209 1045 NaH 2 PO 4 H 2 O 0.29 4.1 20.5 Hepes 10 238 1190 Na Lactate 21.6 368 ul 1840 ul (60% syrup) Phenol Red 1 ul/ml 100 ul 500 ul CaCl 2 2H 2 O* 2.10 29 145 MgCl 2 6H 2 O* 1.5 31 155 *Add last. Check osmolarity (290-310 mOSM). Filter into sterile bottle. Store at 4° C. Media components derived from: Parrish, J. J., J. L. Susko-Parrish and N. L. First. 1985. Theriogenology 24:537. 2 Chemical Components Sigma Catalog Number Abbreviation Name C7902 CaCl 2 *2H 2 O Calcium Chloride-H 2 O H3375 Hepes M2393 MgCl 2 —6H 2 O Magnesium Chloride-6H 2 O P1644 Percoll P0290 Phenol Red P5405 KCl Potassium Chloride S5761 NaHCO 3 Sodium Bicarbonate S5886 NaCl Sodium Chloride L4263 Sodium Lactate (60% syrup) S9638 Na 2 HPO 4 *H 2 O Sodium Phosphate B. Nuclear transfer using somatic cells isolated from semen Using the above techniques, we have found that when a single straw of semen is thawed and put in culture under conditions that will favor the growth of epithelial/fibroblast-like cells, colonies can be detected. Using this protocol, we were able to obtain somatic cells from a straw of bull semen, and use those somatic cells to generate embryos by nuclear transfer. Three replicates of nuclear transfer were performed with three separate Londondale Sperm Cell Lines: 3 Cultured Cleaved % Cleaved Blastocysts % Blastocysts 51 26 51% 9 18% 191 73 38% 37 19% 49 28 57% 10 20% 6 embryos were transferred into three recipients, but no pregnancy was detected. One replicate of nuclear transfer was performed with a Whiteleather Mark Sperm Cell Line. 4 Cultured Cleaved % Cleaved Blastocysts % Blastocysts 53 18 33% 8 15% 6 Embryos were transferred into 3 recipients-1 pregnancy was detected and is still ongoing (approx 67 days-sexed as male). C. Characterization of Sperm Cell Lines Karyotyping Karyotypes were done on both sperm cell lines; images taken and saved. Results indicate that the cells are of bovine origin and have 60 chromosomes. Samples of NT embryos, cell line, semen and extracted DNA were sent to Celera AgGen for DNA analysis. Staining of Semen Cell Line Initial staining of cell lines was performed using alpha tubulin as a general (positive control) marker and Pan Cytokeratin as epithelium marker. Results indicated that there was no staining for the Pan Cytokeratin marker for both concentrations used. Alpha tubulin positive control worked (images not shown). This suggests that the cells are not of epithelial nor endothelial origin, and are probably fibroblasts.