WO2002019811A2

WO2002019811A2 - Generation of transgenic animals using nuclear transfer and oocytes at the germinal vesicle stage

Info

Publication number: WO2002019811A2
Application number: PCT/CA2001/001305
Authority: WO
Inventors: Bin Wang
Original assignee: Nexia Biotechnologies, Inc.
Priority date: 2000-09-06
Filing date: 2001-09-06
Publication date: 2002-03-14
Also published as: AU2001291558A1; WO2002019811A3

Abstract

The invention provides methods for generating transgenic animals using nuclear transfer techniques and oocytes at the germinal vesicle stage.

Description

GENERATION OF TRANSGENIC ANIMALS USING NUCLEAR TRANSFER AND OOCYTES AT THE GERMINAL VESICLE STAGE

Background of the Invention This invention relates to methods for the generation of transgenic animals.

The post-injection rate of integration of DNA into domestic animal embryos, such as cattle, pigs, sheep, and goats is low compared to that of rodents (Wall, Theriogenology pp. 57-68, 1996). The reasons for such low rates are poorly understood. As large numbers of domestic animals are required for embryo transfer, this low DNA integration efficiency makes the production of transgenic large animals difficult. Some studies have suggested that DNA integration into embryos depends on the formation of breaks in the genome (Palmiter et al., Annu. Rev. Genet. 20:465-499, 1986), which is a rare event. More recent research has shown that ligation plays an important role in the generation and reorganization of transgene arrays of injected genes prior to their integration into the genome

(Burdon and Wall, Molecular Reproduction and Development 33:436-443, 1992).

The birth of the cloned lamb Dolly opened a new era for both animal cloning and transgenic animal research and production (Wilmut et al, Nature 385:810-813, 1997). The nuclear transfer (NT) processes have enabled scientists to use genetically modified somatic cells as nuclear donors to create an animal with introduced foreign genes, including cattle. In spite of the rapid advances in animal cloning technologies, including the most recent progress in mice (Wakayama et al., Nature 394:369, 1998), some obstacles still remain. Each step in the process of generating a transgenic animal using nuclear transfer techniques, including cell fusion, oocyte activation, and embryo culture, suffers from low efficiency. Overall, only 1% of reconstructed sheep embryos (Wilmut, supra) and 2-6% of transferred mice clone embryos can give birth to offspring (Wakayama, supra). A large amount of pre- and peri-natal death occurs in all of the nuclear transfer experiments described to date. Further improvements in the efficiency of animal cloning by nuclear transfer will rely on a better understanding of developmental differentiation and reprogramming events, oocyte activation, and embryo culture.

Summary of the Invention

We have invented alternative approaches for introducing DNA into the genome of an animal, through germinal vesicle (GV) stage oocyte reconstruction by nuclear transplantation. Before describing the new methods in detail, it is useful to describe the background science on which the invention depends. In mammalian species, female germ cells migrate into the germinal ridge of the early embryo, and complete a series of mitotic cell divisions. Around the time of birth, the germ cells enter prophase of meiosis I, following a final phase of DNA synthesis. These germ cells, referred to as oocytes, arrest at the diplotene stage of meiosis and are distinguished by a large nucleus, termed the germinal vesicle. Fully grown oocytes are actually arrested at the first meiotic prophase. Completion of the meiotic cell cycle requires (1) oocyte growth, along with proliferation of the surrounding somatic cells, until the oocyte is fully grown; and (2) exposure to a preovulatory gonadotropin surge, which occurs cyclically after puberty. Throughout their growth phase, oocytes develop their competence to resume meiotic maturation, but remain arrested at the GV stage within follicles, until those destined to ovulate respond to gonadotropin stimulation (Yanagimachi, "Mammalian Fertilization," The Physiology of Reproduction, eds. E. Knobil, J. Neill, L.L. Ewing, G.S. Greenwald, C.L. Markert, and D.W. Pfaff, 1998, Raven Press, N.Y. pp. 235-185; Eppig et al, Devel. Biology 164:1-9, 1994). The present invention employs a GV stage oocyte as the recipient of a somatic cell for nuclear transfer. The somatic cell is genetically modified. The reconstructed GV oocyte is then matured by in vitro culture, generating an Mil stage oocyte whose haploid genome is derived from the introduced genetically modified somatic cell nucleus. The oocyte is then activated and the somatic cell pronucleus is removed and fused into a pronuclear stage embryo (zygote) with one of its two pronuclei already removed. The reconstructed zygote is next cultured and transferred to a recipient mother to develop to term.

Accordingly, in a first aspect, the invention features a method for generating a non-human transgenic animal containing a desired gene. The method involves recovering a germinal vesicle stage oocyte from a donor animal; providing a somatic cell containing the desired gene; fusing the somatic cell with the oocyte to form a fused couplet; allowing the couplet to mature; and activating it to produce an activated couplet containing an oocyte-derived pronucleus and a somatic cell-derived pronucleus. The somatic cell-derived pronucleus is then removed from the activated couplet and fused with a pronucleus stage embryo lacking one pronucleus, to form a reconstructed zygote. This reconstructed zygote, or a cleaved embryo, morulae, or blastocyst formed from culturing the zygote, is then transferred into a recipient animal, and developed to term to produce a transgenic animal containing a desired gene.

In a preferred embodiment of the invention, the germinal vesicle stage oocyte contains a nucleus or is enucleated. The somatic cell may be in G_ls G₂, or M phase of the cell cycle. The animal is preferably a mammal, more preferably a ruminant, for example, a sheep, a goat, or a cow, or it may be a pig or a rabbit. The somatic cell and germinal vesicle stage oocyte can be derived from the same animal, or from different animals, and may be from the same or different species. The couplet can be activated by parthenogenetic activation or by sperm-mediated activation.

By "transgene" is meant any piece of nucleic acid that is inserted by artifice into a cell, or an ancestor thereof, and that becomes part of the genome of the animal which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic animal, or may be a gene homologous to an endogenous gene of the animal. By "transgenic" is meant any cell which includes a nucleic acid sequence that has been inserted by artifice into a cell, or an ancestor thereof, and that becomes part of the genome of the animal which develops from that cell. Preferably the transgenic animal is a transgenic mammal (e.g., a rodent or ruminant). In addition, the nucleic acid (transgene) is preferably inserted by artifice into the nuclear genome.

By a "germinal vesicle stage oocyte" is meant an oocyte whose nucleus is arrested in a postsynaptic stage of meiotic prophase I.

By a "somatic cell" is meant any cell that does not contribute to the production of a gamete.

By a "cleaved embryo" is meant a zygote that has undergone a number of cell divisions and is in the process of developing into a morula or a blastocyst.

By an "elite animal" is meant an animal that is highly valuable in terms of genetic traits in productivity (for example, milk yield or protein production), reproduction, or disease resistance. An elite animal may be an animal that contains a foreign gene encoding a protein of commercial value. An elite animal may also be an endangered species.

The methods of the invention are more efficient than standard microinjection and animal cloning by nuclear transfer techniques. In addition, the invention provides superior elite animal reproduction for the following reasons.

1) Because the introduced somatic cell nucleus goes through oocyte maturation, fertilization, activation, and pronuclear stage and early embryo development, the donor cell genome has more chances to be completely reprogrammed. In comparison, in standard nuclear transfer techniques, the somatic cell nucleus is introduced into the oocyte later, for example, after the oocyte has matured. The introduction of a somatic cell nucleus into a GV stage oocyte will lead to higher survival rates of the embryos and healthier offspring after embryo transfer. 2) When oocyte activation is carried out by an intact sperm, the development of the reconstructed embryos will be superior to that from artificial activation used in other animal cloning by nuclear transfer techniques.

3) The donor cell's genome can be modified through standard procedures, so that a foreign gene can be introduced into the offspring developed from the reconstructed oocytes. If the foreign gene integration site in the donor cell is single, the transgenic rate should be 50% of the offspring, based on the fact that a foreign gene has a fifty percent chance of being expelled into the first polar body. If the integration sites are multiple, the transgenic rate will be 100%. Through this approach, the animal's genome can be precisely modified through gene target technology, which cannot be achieved by traditional microinjection methods.

4) In animal genetic breeding systems, early breeding is often required to shorten the generation interval. By using the technology described herein, for example, by using a somatic cell from the fetus of an elite male or female animal, offspring can be conceived in a foster mother even when the elite animal is still in the fetal stage, thus decreasing the interval between generations.

Brief Description of the Drawing The Figure is a schematic representation of the steps involved in producing a transgenic embryo through GV oocyte reconstruction according to the invention.

Detailed Description Referring to the Figure, on the left side of the schematic, GV stage oocytes are stripped of cumulus cells. A somatic cell carrying the foreign gene is introduced into the perivitelline space at the opposite position of the germinal vesicle. Following electric fusion and maturation culture, the oocytes are subjected to artificial parthenogenetic activation through a procedure that could lead to second polar body release (if donor cells are synchronized at G₂ or M phase during nuclear transfer) or no second polar body release (if donor cells are at G₀ or Gi phase). The somatic cell-derived pronucleus is then separated from the reconstructed oocyte based on its pronuclear location in the cytoplasm; the pronucleus near the slit on the zona pellucida, which is created during cell transfer, is presumed to be of somatic cell origin. The separated pronucleus is transferred and fused into a pronucleus stage embryo whose female or male pronucleus has been removed previously (depicted on the right side of the schematic). This reconstructed zygote is cultured in vitro to develop to a suitable embryo stage, and transferred into a foster mother for further development.

Techniques for carrying out each method of the invention are now described in detail, using particular examples. These examples are provided for the purpose of illustrating the invention, and should not be construed as limiting.

Materials and Methods

1. Oocyte Collection and Culture

Bovine ovaries were collected from a local abattoir and transported to the laboratory in a thermos containing D-PBS (Gibco, Grand Island, NY) at 20-25°C. Within 1-2 hours after ovary arrival, the oocytes (in the form of cumulus-oocyte complexes) were aspirated from 2- to 5-mm ovary follicles, and cultured in maturation medium (TCM199 (Gibco) supplemented with 10% heat inactivated fetal calf serum (Immunocorp), 0.1% gentamicin (Gibco), 5 μg/ml luteinizing hormone (Noble Laboratory), 0.5 μg/ml follicle-stimulating hormone (Noble Laboratory), 1 μg/ml estradiol (Sigma), and 22 μg/ml pyruvate (Gibco)). The culture environment was 5% C0₂ and 95% humidified air at 38.5°C. One to 1.5 hours later, the cumulus cells were removed to denude the oocytes, by vortexing the cumulus-oocyte complex for 1.5 minutes. 2. Germinal Vesicle Stage Oocyte Enucleation

The germinal vesicle (GV) in each oocyte was located under a Nomarski optic 400X field. A 20-25 μm slit was made on the zona pellucida near the GV. A small amount of cytoplasm along with the GV was squeezed out of the slit by pushing the oocytes with a microglass needle. Fifteen to 20 minutes later, the karyoplasts were pinched off by pipetting the oocytes through capillary glass tubing, producing enucleated oocytes. The enucleated oocytes were temporarily cultured in droplets of Emcare embryo flushing media containing 1% FCS on a 37°C warm plate, until transfer of the somatic donor cell.

3. Somatic Donor Cell Transfer and Electrofusion

A bovine fetal fibroblast cell line was maintained in culture using standard cell culture methods. On the day of somatic cell transfer, the fibroblast monolayer was trypsinized and resuspended in Emcare containing 1% FCS. This cell suspension solution was transferred into the perivitelline space (PVS) of enucleated oocytes or non-enucleated GV oocytes at the opposite position of the GV, through microinjection with a 15 μm beveled glass pipette. The cells were gently pushed against the oocyte membrane in order to make tight contact between the cell membranes. These somatic cell and oocyte couplets were then cultured in micromanipulation drops of maturation medium (TCM199 media containing 25 mM HEPES supplemented with 10% FCS, 0.01 units/ml bFSH, 0.01 units/ml bLH, 1 μg/ml E₂ (estradiol), 22 μg/ml sodium pyruvate, and 1 μg/ml gentamicin) until electrofusion. The micromanipulation drops were maintained at room temperature during micromanipulation and the electrofusion procedure. The electrofusion medium was 0.25 M D-Sorbital (Sigma) containing 100 μM CaOAc (Sigma), 0.5 mM MgOAc (Sigma), and 0.1% BSA (Fatty acid free, Sigma). Fusion pulse parameters were maintained at a constant single 1.84 KV/CM pulse for 15 μsec. The time that the electrofusion occurred varied from one experiment to another, ranging from 4 to 7 hours post-oocyte culture. Fused couplets were separated from non-fused couplets and cultured in maturation medium, as described above for 16 to 22 hours to mature the oocytes.

4. Artificial Activation Artificial activation of the matured oocytes was performed according to the method of Susko-Parrish et al., Dev. Biol. pp. 729-730 (1995). The oocytes were exposed to the calcium ionophore, ionomycin (5 μM, Cal Biotech, La Jolla, CA) in Emcare supplemented with 1 mg/ml BSA, for 5 minutes and then were washed for 5 minutes in Emcare supplemented with 30 mg/ml BSA. The matured oocytes were then cultured in BECM containing 3 mg/ml BSA and 0.2 μM

4-Dimethylaminopyridine (DMAP; Sigma) for 4.5 hours at 38.5°C under 5% C0₂. The oocytes were then washed and cultured in 100 μl droplets of BECM containing 3 mg/ml BSA, and were covered by pre-equilibrated light weight oil, until they developed to the pronuclear stage.

5. Preparation of Recipient Zygotes

The bovine zygotes were derived by in vitro maturation and in vitro fertilization. The procedure is briefly described as follows:

The oocytes were aspirated from 2-5 mm size follicles from the ovaries obtained from a local slaughter house. The oocytes with the intact cumulus cell mass and homogenous cytoplasm were selected for in vitro maturation. The maturation medium was described as above. After maturation, oocytes were inseminated by freeze-thawed bull spermatozoa. At 17-19 hours post insemination, zygotes were stripped of cumulus cells and centrifuged at 15,600 g for 6 minutes to stratify the cytoplasm for visualization of pronuclei.

6. Pronuclear Exchange

The pronuclear stage embryos from the NT procedure and the recipient zygotes were centrifuged at 15,600 g for 6 minutes in order to visualize the pronuclei under Nomarski contrast optics. A 45 μm beveled pipette was inserted into the PVS of the NT-derived oocyte to aspirate the pronucleus near the slit on the zona pellucida. By a combination of two criteria, the somatic cell-derived pronucleus can be recognized. First, if the cytoplasm did not rotate during maturation, the somatic cell-derived pronucleus should be closest to the slit that was created during the GV oocyte reconstruction. In addition, one of the two pronuclei often appears smaller than the other one. If the somatic cells are at G₀ or Gi during GV oocyte reconstruction, the DNA content of the somatic cell nucleus is only 50% that of the germinal vesicle-originated pronucleus. After activation, the size of the somatic cell-derived pronucleus should be smaller than the germinal vesicle-originated pronucleus germinal vesicle.

The donor pronucleus was then transferred into the PVS of the pronuclear recipient zygote whose female pronucleus was previously removed. The pronuclear exchange was completed using the same electrofusion parameters described above. The reconstructed zygotes were cultured in S OF medium at 38.5°C under 5% C0₂, 7% 0₂, and 88% N₂ for 7 days to examine its in vitro development.

7. Chromatin analysis Oocytes and nuclear transfer embryos were fixed in a glutaraldehyde and

Triton X 100 solution and stained with Hoechst 33342. The samples were mounted on glass slides, and the nuclei and chromatin were evaluated under an epifluorescence microscope.

8. Chromosome analysis

Metaphase oocytes were first removed of zona pellucida by digestion in a solution containing Emcare and 0.2% pronase E (from streptomyces griseus, Sigma), followed by hypotonic treatment in 40% fetal calf serum in deionized water for 7 minutes. The oocytes were then fixed with acetic acid, methanol, and _*

water (in a 1 :5:4 ratio) for 2-10 minutes on ice. The oocytes were then individually fixed again with acetic acid and methanol (in a 1:3 ratio) until the cytoplasm became transparent. A single oocyte was then dropped onto a glass slide and stained in 0.02% Giemsa for 4 minutes. The chromosome spreads were then examined under a microscope.

Transfer of Somatic Donor Cells into Enucleated GV Stage Oocytes and Maturation

The GV stage oocytes were denuded and enucleated, as described above. The somatic bovine fibroblast cells were transferred and fused into the cytoplasm of each ova, also as described above. The cell cycle of the donor cell was not synchronized in this experiment. Approximately 16 and 22 hours after oocyte isolation, the reconstructed oocytes were stained and evaluated for nuclear maturation. This was done by staining the oocytes with Hoechst 33342, and observing the morphology of the chromatin using an epifluorescence microscope. The nucleus can be readily categorized to be GV, MI, Mil, or interphase between MI and MIL As a control, denuded oocytes were cultured in IVM medium under the same conditions as the reconstructed oocytes. The experiment was replicated three times. The results from this study are shown in Table 1. A high GV enucleation rate (95%) was obtained in this study. When the reconstructed oocytes were cultured for 16 hours, 100% of the transferred somatic cell nuclei became condensed; by 22 hours, the majority of oocyte nuclei (71%) still remained condensed, with only a few reaching the MI, MI-MII, or Mil stage. In contrast, 71% of the control denuded oocytes developed to the MI-MII stage at 14 hours, and 88% reached the Mil stage at 22 hours. Table 1. Cell Cycle Status After Transfer of Bovine Fibroblast Cells into Enucleated GV Oocytes and Maturation

NT= nuclear transfer

Rep. = of experiment replications

Transfer of Donor Cells into Non-enucleated GV Stage Oocytes and Maturation

The GV stage oocytes were denuded, as described above, but they were not enucleated. A bovine fibroblast cell was transferred and fused into the cytoplasm of each ovum, also as described above. Again, the cell cycle of the donor cells was not synchronized. The level of maturation was compared to that of denuded oocytes cultured in IVM medium under the same conditions as the reconstructed oocytes. At 16 and 22 hours of maturation culture after oocyte isolation, the reconstructed oocytes were evaluated for their level of maturation, as described above. The experiment was replicated three times.

This study was similar to the previously described study, with the exception that the recipient GV oocytes were not enucleated. The data in Table 2 show that 92-93% of the reconstructed oocytes contained two first polar bodies at 16 hours and 22 hours of culture. One of the two first polar bodies resulted from the meiosis cleavage of the transferred somatic cell nucleus. Most of the reconstructed oocytes (93%) completed nucleus maturation within 22 hours of culture. At 16 hours post maturation culture, the reconstructed oocyte group demonstrated in a similar maturation rate as the control group (79% of reconstructed oocytes versus 71% of control oocytes).

Table 2. Cell Cycle Status After Transfer of Bovine Fibroblast Cells into Non-enucleated GV Oocytes and Maturation

NT=nuclear transfer PBs= polar bodies

Chromosome Analysis of GV-NT Derived Mil Oocytes to Demonstrate the Meiosis Separation of Somatic Cell Chromosomes

Non-enucleated bovine cumulus cells and fetal fibroblast cells, as described in the previous section, were starved for 3-4 days to synchronize the cell cycles at the Gr/Gi stage. Chromatin condensation in the nuclei of cells at Gr Gi phase can produce chromosomes with a single chromatid. Therefore, in the Mil stage oocytes derived from GV-NT, the chromosomes from somatic cells can be distinguished from chromosomes of the oocyte itself. Mature oocytes and the Mil oocytes fused with Go/Gi cells were used as controls to evaluate the chromosome morphology. The experiment was replicated 3 times.

The results of this study are given in Tables 3 and 4. After cell fusion and maturation, most of the GV-NT couplets (89%) released two first polar bodies (Table 3). One of the two polar bodies resulted from the cleavage of the somatic cell nucleus. To confirm the meiosis cleavage of the transplanted somatic cell nucleus, the karyotypes of oocytes resulting from GV-NT were analyzed (Table 4). Of the analyzable spreads, 58% were determined to be of somatic cell origin and showed evidence of meiotic cleavage.

Table 3. Cell Fusion and Maturation

NT=nuclear transfer PB=ρolar body

Table 4. Chromosome Analysis

I

I- » en

In Vitro Fertilization or Parthenogenetic Activation of GV Reconstructed Oocytes

In order to examine the competence of somatic cell-derived Mil nuclei for further development, the reconstructed oocytes were activated by either spermatozoa, through standard IVF procedures, or by artificial activation with calcium ionophore combined with 6-DMAP.

Since a high maturation rate was obtained using non-enucleated GV oocytes as NT cytoplasmic recipients, in the following experiments non- enucleated GV oocytes were designated for parthenogenetic activation by in vitro fertilization using spermatozoa. At about 18 hours post-insemination, the oocytes were stained to evaluate the pronuclear development. Three replicates of this experiment were conducted.

As shown in Table 5, after fusion and culture in maturation medium for 22 hours, 78% of the oocytes matured. All fused oocytes were next subjected to in vitro fertilization. Although the denuded oocytes had low fertilization rates, both in the control and NT groups, some of the oocytes showed three pronuclei (15%) or more than three pronuclei (2%) (one of the three pronuclei was presumed to be of somatic cell origin). Those oocytes with two pronuclei in the NT group and one pronucleus in the control group may have resulted from spontaneous parthenogenetic activation.

Table 5. Status of Embryos After Transfer of Bovine Fibroblast Cells into Non-enucleated GV Oocytes, Maturation, and IVF

NT= nuclear transfer

2PB1= two first polar bodies

Due to the low in vitro fertilization rate of the denuded oocytes, the competence of the somatic cell nucleus developing into a pronucleus could not be examined accurately using standard IVF procedures. Therefore, artificial activation was carried out using A23187 and 6-DMAP. Briefly, the oocytes were first treated with A23187 for 5 minutes and then treated with 6-DMAP for 4 hours or overnight. Treatment with A23187 is done to increase the calcium in the cytoplasm, which is well recognized as a signal to activate the cell cycle. 6- DMAP is used to inhibit the activity of maturation promoting factor (MPF), leading to full activation of an oocyte. The oocytes with two first polar bodies were chosen for activation treatment after 22 hours of maturation culture. The development of the nuclei into pronuclei was then measured 18 hours post- activation.

The results of this study (Table 6) demonstrated that in 69% of the NT- derived GV stage oocytes, the metaphase nucleus derived from the somatic cell could develop into a pronucleus with normal morphology along with the oocyte metaphase nucleus. Some of the oocytes in the NT group yielded one (10%), three (7%), and four (7%) pronuclei, respectively. In the control group, the majority of oocytes activated by A23187 combined with 6-DMAP developed one pronucleus (93% of the oocytes).

Table 6. Artificial Activation and Pronuclear Development of NT Oocytes

NT= nuclear transfer PN= pronucleus/pronuclei

Pronuclear Exchange Between Nuclear Transfer Derived Oocytes and IVF- Derived Zygotes

The competence of the somatic cell-derived pronucleus was further investigated. A female pronucleus in the fertilized recipient oocyte was replaced by a somatic cell- derived pronucleus, and the reconstructed zygotes were cultured in Gl + G2 medium for 7 days. The zygotes generated by pronuclear exchange within fertilized oocytes were used as controls. The results of this study are given in Table 7. The initial cleavage rate of GV-NT-derived oocytes was similar to the control (74% for the GV-NT-derived oocytes versus 80% for the control oocytes). A small proportion of zygotes (4%) developed to the blastocyst stage in the GV- NT group. The pronucleus exchange micromanipulation procedure does not appear to be harmful to embryo development, since the NT control group had a 50% blastocyst development rate.

Table 7. Development of Zygotes Produced by Pronuclear Exchange

PN3

GV-NT= GV stage oocytes and nuclear transfer PN= pronuclear exchange

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A method for generating a non-human transgenic animal containing a desired gene, said method comprising: (a) recovering a germinal vesicle stage oocyte from a donor animal;

(b) providing a somatic cell containing said desired gene;

(c) fusing said somatic cell with said oocyte to form a fused couplet;

(d) allowing said couplet to mature;

(e) activating said matured couplet to produce an activated couplet containing an oocyte-derived pronucleus and a somatic cell-derived pronucleus;

(f) removing said somatic cell-derived pronucleus from said activated couplet;

(g) fusing said somatic cell-derived pronucleus with a pronucleus stage embryo lacking one pronucleus to form a reconstructed zygote; (h) transferring said reconstructed zygote, or a cleaved embryo, morulae, or blastocyst formed from culturing said zygote, into a recipient animal; and (i) allowing said reconstructed zygote, cleaved embryo, morulae, or blastocyst to develop to term.

2. The method of claim 1, wherein said germinal vesicle stage oocyte contains a nucleus.

3. The method of claim 1, wherein said germinal vesicle stage oocyte is enucleated.

4. The method of claim 1, wherein said somatic cell is in G_1? G₂, or M phase of the cell cycle.

5. The method of claim 1, wherein said animal is a ruminant, a pig, or a rabbit.

6. The method of claim 1, wherein said somatic cell and said germinal vesicle stage oocyte are derived from the same animal or different animals.

7. The method of claim 1, wherein said somatic cell and said germinal vesicle state oocyte are derived from different animals.

8. The method of claim 1, wherein said somatic cell and said germinal vesicle stage oocyte are derived from the animals of the same species.

9. The method of claim 1, wherein said somatic cell and said germinal vesicle stage oocyte are derived from the animals of different species.

10. The method of claim 5, wherein said ruminant is a goat.

11. The method of claim 5, wherein said ruminant is a sheep.

12. The method of claim 5, wherein said ruminant is a cow.

13. The method of claim 1, wherein said activation is parthenogenetic activation.

14. The method of claim 1, wherein said activation is sperm-mediated.