WO2000065035A1

WO2000065035A1 - Telophase enucleated oocytes for nuclear transfer

Info

Publication number: WO2000065035A1
Application number: PCT/CA2000/000483
Authority: WO
Inventors: Lawrence C. Smith; Vilceu Bordignon
Original assignee: Universite De Montreal
Priority date: 1999-04-28
Filing date: 2000-04-27
Publication date: 2000-11-02
Also published as: JP2002542779A; CA2370385A1; BR0010588A; AU4281500A; EP1179053A1; AU777799B2; MXPA01010905A; KR20020008398A; NZ515159A

Abstract

The present invention relates to an improved method for obtaining an enucleated host oocyte for transferring nuclei from embryonic, germinal and somatic cells with the objective of cloning or multiplying mammals, and to a method of reconstituting a non-human embryo.

Description

TE OPHASE ENUCLEATED OOCYTES FOR NUCLEAR TRANSFER

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The present invention relates to an improved method for obtaining an enucleated host oocyte for transferring nuclei from embryonic, germinal and somatic cells with the objective of cloning or multiplying mammals, and to a method of reconstituting an animal embryo. (b) Description of Prior Art

The technique of nuclear transfer has been widely used to multiply embryos by transferring blastomere nuclei from early-stage embryos into enucleated oocytes. This technique enables an increase in the yield of embryos produced from parents of top genetic value, enabling to accelerate the annual genetic gain within an animal population. Nuclear transfer has also been used with nuclei from cell lines derived from embryonic (Campbell et al., 1996, Na ture 380: 64-66) , .fetal and adult tissue ( ilmut et al., 1997, Na ture 385:810-813) . By using nuclei from an unlimited source, nuclear transfer from cell lines enables not only the production of a larger number of genetically identical offspring but also an opportunity for modifying the genetic characteristic of cells in vi tro prior to the production of live offspring, enabling the production of transgenic mammals. Moreover, the use of cells from adult animals for nuclear transfer, either directly or through previous in vi tro passage, enable the multiplication (cloning) of animals of known phenotypes.

Basically, the nuclear transfer technique requires a donor nucleus to provide the genetic material of choice and a host oocyte to provide the cytoplasm that plays a role in reprogramming the nucleus to support embryo development. With the nuclear and cytoplasm sources in hand, three main steps are required to reconstruct an oocyte by nuclear transfer. First, host oocytes need to be enucleated to remove all nuclear genetic material. This step is usually performed by microsurgical removal of the chromosomes from either a metaphase plate or pronuclei. Second, donor nuclei need to be introduced into the oocyte (nuclear transfer) . This step is normally obtained by fusing the membranes of the nuclear donor cell and the host oocyte. However, nuclear transfer can also be obtained by traversing the oocytes plasma membrane and microinjecting the nucleus directly into the host cytoplasm. Finally, non-activated host oocytes need awakening from their meiotic arrest (oocyte activation) . This step can be achieved by exposing the oocyte to a physical stimulus, such as temperature changes or an electric shock, or exposing the oocyte to chemical agents, such as ethanol or exogenous calcium. The order in performing each of the steps above can vary in different situations and may have an important effect on the ability of the reconstructed oocyte to undergo further development .

In mice, oocyte enucleation was performed after fertilization by visualizing and removing the pronuclei by microsurgery. This enucleation technique is less efficient in other mammals due to the higher density of cytoplasm resulting in poor visualization of pronuclei. Moreover, attempts to use pronuclear-stage enucleated oocytes led invariably to poor developmental rates when using cleavage stage blastomeres as nuclear donors. Improved development after nuclear transfer was achieved initially in sheep (Willadsen, S . 1986, Na ture 320: 63-65) and later in other mammals using host oocytes that had not been activated at the time of fusion. However, a problem remained that metaphase stage chromatin cannot be visualized easily by microscopy in most mammals. Willadsen (Willadsen, S.1986, Na ture 320: 63-65) proposed an enucleation procedure in which sheep oocytes were blindly divided into halves either containing or not the first polarbody. To avoid a large loss of cytoplasm during enucleation, this procedure was later improved by using a DNA vital stain (Bisbenzimide; Hoechst) and ultraviolet (UV) irradiation to check whether the Mil plate after removal of small portions of cytoplasm. The most common procedure of oocyte enucleation is to expose secondary oocytes to bisbenzimide, blindly remove a cytoplasmic fragment surrounding the first polarbody and then expose the oocyte to UV to ascertain whether enucleation was correctly performed. On average this procedure correctly enucleates between 60 to 80 percent of oocytes. Another possible limitation of this procedure is that oocytes are exposed both to UV irradiation and Hoechst 33342 that have been shown to have detrimental effects on the cytoplasm (Smith, L. 1993 J. Reprod. Fert . 99:39-44) .

As mentioned above, host oocytes are able to support better development after nuclear transfer when compared to pronuclear-enucleated host zygoteslt has already been shown that Mil-stage enucleated oocytes either aged or activated before fusion support better development. The problem of using young non-activated oocytes is caused by incompatibilities between the cell cycle stages of the nuclear donor cell and the host cytoplasm. Metaphase arrested secondary (Mil) oocytes have high levels of a Maturation Promoting Factor

(MPF) , a cellular activity that is responsible for maintaining the chromatin condensed without a nuclear envelop. When blastomere interphase-stage nuclei containing decondensed chromatin are introduced into an Mil oocyte, MPF leads to a rapid breakdown of the nuclear membrane and premature chromosome condensation (PCC) . However, PCC is believed to be detrimental only when induced during the DNA synthesis stage (S-phase) of cell cycle. This is particularly problematic when using donor nuclei from blastomeres since these undergo S-phase for most time in between cell divisions. On the other hand, enucleated oocytes that have been activated or aged before fusion to nuclear donor cells have lower levels of MPF and, therefore, do not cause PCC.

With the exception of blastomeres, most other cell types have longer gaps both before (Gl-phase) and after (G2-phase) the S-phase and, therefore, are less susceptible to the harmful effects of S-phase PCC when fused to a Mil oocytes. Because high MPF levels cause the breakdown of the nuclear membrane, Mil stage host oocytes are believed to facilitate interactions between donor nuclei and putative oocyte cytoplasmic 'factors' required for reprogramming the chromatin of nuclei derived from cells further advanced in differentiation. Several examples in the literature report on the advantages of passaging further differentiated donor nuclei in non-activated Mil oocytes before activating the reconstructed oocyte. In cattle, nuclei from an embryonic cell line supported significantly higher yield of blastocyst development and more 30d pregnancies when fused to enucleated oocytes 4 h before activation. In mice, significantly more embryos reconstructed with cumulus cell nuclei developed to the blastocyst stage by exposing the donor nucleus to Mil cytoplasm for between 1 and 6 h before activation

(Wells et al. 1999, Biol . Reprod. 60:996-1005) .

Moreover, no fetal development or live offspring was obtained when using with simultaneous activation and fusion. Furthermore, other reports using differentiated cell lines have used host oocytes that were either activated after or concurrently with introducing the donor nucleus (Cibelli et al. 1998, Na ture Biotechnol . 16:642-646; Wilmut et al . 1997, Na ture 385:810-813) . Therefore, the prevalent theory in the field of cloning by nuclear transfer is that a period of reprogramming in the cytoplasm of an inactivated oocyte is required to obtain success when using donor nuclei from cells other than embryonic blastomeres.

It would be highly desirable to be provided with an improved method for obtaining an enucleated host oocyte for transferring nuclei from embryonic, germinal and somatic cells with the objective of cloning or multiplying mammals.

It would be highly desirable to be provided with an improved method of reconstituting an animal embryo .

SUMMARY OF THE INVENTION

The present invention described below is contrary to current knowledge in that we are teaching use of an activated oocyte as recipient for nuclei derived from cells from embryonic and somatic cell lines.

One aim of the present invention is to provide an improved method for obtaining an enucleated host oocyte for transferring nuclei from embryonic, germinal and somatic cells with the objective of cloning or multiplying mammals.

Another aim of the present invention is to provide an improved method of reconstituting a non- human embryo.

In accordance with the present invention there is provided a method of preparing an enucleated host oocyte for transferring nuclei from embryonic, germinal or somatic cells, which comprises the steps of: a) activating oocyte by artificial means; and b) enucleating the activated oocyte when the activated oocyte is undergoing the expulsion of a second polarbody or when the activated oocyte has recently expelled second polarbody (Tel- II); and c) transferring nuclei from embryonic, germinal or somatic cells into the enucleated oocyte of step b) , wherein embryonic cells are cultured prior to nuclei transfer.

The germinal or somatic cells are cultured prior to nuclei transfer. The oocyte of step a) has a first polarbody and the artificial means is chemical means, such as ethanol or ionomycin.

Step b) may be performed after oocytes are cultured for a period of time sufficient to allow for extrusion of a second polarbody.

Step b) may be performed with oocytes in a medium with cytosqueletal inhibitors.

Step b) may be effected by microsurgically removing the second polar with about one tenth of the cytoplasm surrounding the second polarbody.

The preferred oocyte is a secondary (M-II) oocyte .

In accordance with the present invention, there is provided a method of reconstituting a non-human embryo, which comprises the steps of: a) activating oocyte by artificial means; b) enucleating the activated oocyte when the activated oocyte is undergoing the expulsion of a second polarbody or when the activated oocyte has recently expelled second polarbody (Tel- II); c) transferring a diploid nucleus in the enucleated oocyte to obtain a reconstructed oocyte with a diploid chromosomal content; and d) culturing in vi tro the reconstructed oocyte and/or transferring the reconstructed oocyte into a reproductive tract of a suitable surrogate mother to enable development into a non-human embryo.

In accordance with the present invention, there is provided a method for production of a transgenic non-human embryo, which comprises the steps of: a) transfecting cultured cells selected from the group consisting of embryonic, germinal and somatic cells with a desired DNA construct; b) activating oocyte by artificial means; c) enucleating the activated oocyte when the activated oocyte is undergoing the expulsion of a second polarbody or when the activated oocyte has recently expelled second polarbody (Tel-II) ; d) transferring a diploid nucleus extracted from the transfected cells of step a) in the enucleated oocyte to obtain a reconstructed oocyte with a diploid chromosomal content; and e) culturing in vi tro the reconstructed oocyte and/or transferring the reconstructed oocyte into a reproductive tract of a suitable surrogate mother to enable development into a non-human embryo. The non-human embryo may develop into a non- human animal . BRIEF DESCRIPTION OF THE DRAWING

Fig. 1 illustrates a schematic protocol of the technique of telophase enucleation for nuclear transfer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of producing embryos by nuclear transplantation from embryonic, germinal and somatic cells lines. Nuclear transfer procedures have invariably initiated with the enucleation of host oocyte. The enucleation procedure is followed by one of the following: (a) activation followed by fusion; (b) concurrent activation and fusion; or (c) fusion followed by activation. Whereas the procedure in which oocytes are (a) enucleated, activated and then fused is used mostly for embryonic blastomeres, most techniques applied for further differentiated donor nuclei use the procedure where oocytes are enucleated, (b) fused and activated concurrently or (c) fused and later activated. Although the different approaches in the nuclear transfer procedure have been described previously (U.S. Patent No. 4,994,384; U.S. Patent No. 5,057,420; U.S. Patent No. 5,843,754 and International Patent applications Nos. PCT/GB96/02098, PCT/US98/00002,

PCT/US98/12800, PCT/US98/12806, and PCT/US97/12919) , the present invention describes a sequence of steps in the nuclear transfer procedure that is novel (Fig. 1) .

As illustrated in Fig. 1, Step 1 involves the activation of secondary (M-II) oocytes by artificial means. Step 2 is performed shortly after activation when the oocyte is undergoing the expulsion or recently expelled the second polarbody (Tel-II). Step 3 relates to the transfer of a nucleus from any source with the purpose of reconstructing the oocyte with a diploid chromosomal content.

Step 1 (oocyte activation)

Oocytes are obtained either in vivo or in vi tro and cultured in maturation medium. After maturation, oocytes are denuded of cumulus cells and those with a first polarbody are parthenogenetically activated by chemical means using ethanol or ionomycin. After activation, oocytes are cultured for a few hours to allow for extrusion of the second polarbody. Step 2 (oocyte enucleation)

After activation, oocytes can be placed in medium with cytosqueletal inhibitors to facilitate microsurgery. Only oocytes with a second polarbody extruded or partially extruded are used. Approximately one tenth of the cytoplasm surrounding the second polar body is microsurgically removed with the second polarbody. Step 3 (nuclear transfer) After enucleation, a single cell containing a diploid nucleus is introduced into the enucleated oocyte either by cell fusion or microinjection (nuclear transfer) . The reconstructed oocyte is then cultured in vitro and/or transferred into the reproductive tract of a suitable surrogate mother to enable further development .

The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope. EXAMPLE 1 Telophase Enucleation

Follicles with 2 to 8 mm diameter were aspirated from bovine slaughterhouse ovaries. Oocytes with a homogeneous cytoplasm and several layers of cumulus cells were selected and placed in maturation within 1 h from follicular aspiration. At 28 h after maturation oocytes were denuded of cumulus cells and those with a first polarbody were used in the experiment. Oocytes were exposed to 7% ethanol for 5 min, washed and placed in maturation medium for different periods. At 1 h before microsurgery, oocytes were placed in cytochalasin B and positioned for micromanipulation. Oocytes undergoing extrusion or already with extruded second polarbodies had 10% of their cytoplasmic volume removed together with the second polarbody. After microsurgery, oocytes were fixed in 10% formalin, stained with 5 μg Hoechst 33342 and observed under UV epi-fluorescence . Oocytes without any chromatin were considered successfully enucleated. Most oocytes were successfully enucleated when micromanipulated at the times examined (Table 1) . Because the efficiency of this enucleation technique is high, checking of oocytes with DNA stain and UV light is not necessary. Significantly lower percentages of enucleation was obtained when blindly removing using the position of the first polarbody to aspirate 30% of the surrounding cytoplasm in oocytes at metaphase (59%) at 24 h from the beginning of in vi tro maturation. Table 1

Successful telophase enucleations as performed at different times after exposure to a stimulus to parthenogenetically activate secondary oocytes

Time after activation

3 h 4 h 5 h Total

Number manipulated 37 38 43 118 Successful enucleation 36 37 40 113 (%) (97%) (97%) (93%) (96%)

Example 2

Nuclear transfer with orula-stage blatomeres

Bovine secondary oocytes were matured in vi tro and enucleated using the technique described above (telophase enucleation) . Morula-stage embryos were disaggregated and individual blastomeres were inserted into the perivitelline space of enucleated oocytes. Fusion between the membranes of blastomeres and oocytes was obtained with an electric pulse that causes fusion between the membranes of the donor and recipient cells. The electrical parameters used were double 60 μsec pulses of 1.5 KVolts per cm. After fusion the embryos were cultured for 7 days in the presence of Menezo's B2 medium supplemented with 10% fetal calf serum.

Table 2

Fusion and development of bovine oocytes reconstructed with nuclei from morula-stage blastomeres recovered 5 days after IVF

Number Fused Blastocyst No. nuclei

Telophase II 215 129 49 126111

(%) (58%) (38%)

Metaphase II 248 151 24 84±9

(%) (60%) (16%) Example 3 Nuclear transfer with non-starved bovine ES cells

Bovine embryo stem (ES)-like cells were obtained from day 8 blastocyst stage embryos produced entirely in vi tro . ICMs were platted onto mitomycin- inactivated mouse fibroblasts. Established ES-like lines were disaggregated by short exposure to trypsin using a narrow pipette. Isolated cells were placed in the perivitelline space of enucleated oocytes and exposed to an electric pulse that causes fusion between the membranes of the donor and recipient cells. The electrical parameters used were double 100 μsec pulses of 1.5 KVolts per cm. Electrical stimulation was performed as soon as possible after placing the nuclear donor cell in the perivitelline space to obtain better fusion results. After fusion the embryos are cultured for 7 days in the presence of Menezo's B2 medium supplemented with 10% fetal calf serum.

Table 3

Fusion and development of bovine oocytes reconstructed with nuclei from ES-like cells exposed to 5% of FCS

Number Fused Cleaved Blastocyst

Telophase II 38 11 5 3

(%) (30%) (45%) (27%) Metaphase II 33 12 2 1

(%) (36%) (17%) (8%)

Example 4 Nuclear transfer with serum-starved bovine ES cells

Bovine embryo stem (ES)-like cells were cultured in medium with 0.5% FCS for 5 days before micromanipulation. As described above, ES-like cells were disaggregated, placed in the perivitelline space of enucleated oocytes and exposed to an electric pulse to cause fusion between the membranes of the donor and recipient cells. After fusion the embryos are cultured for 7 days in the presence of Menezo's B2 medium supplemented with 10% fetal calf serum.

Table 4

Fusion and development of bovine reconstructed with nuclei from bovine ES-like cells exposed (starved) to low concentrations (0.5%) of FCS Number Fused Cleaved Blastocyst

Telophase II(%) 38 13(34%) 3(23%) 2(27%)

Metaphase II(%) 42 13(31%) 4(31%) 1(15%)

Example 5

Nuclear transfer with starved and non-starved bovine fetal fibroblasts

Bovine fetal fibroblast cells were recovered form day 50 fetuses and passaged in medium D-MEM with 10% FCS. Non-starved fibroblast cells were recovered during growth at 2 days after passaging. Serum starved cells were exposed to medium with 0.5% serum for 5 days before NT. NT was performed as described above.

Table 5

Fusion and development of bovine reconstructed with nuclei from bovine fetal fibroblast cells exposed for 5 days to low concentrations (0.5%) of FCS (starved) or to 5% FCS for 20 h after seeding (non-starved)

Serum starved Non-starved

Number Fused Blast. Number Fused Blast.

Telophase II 69 52 2 105 67 9 (%) (75%) (4%) (64%) (13%)

Metaphase II 60 39 9 114 92 12 (%) (65%) (24%) (81%) (13%) Example 6

Nuclear transfer with starved and non-starved bovine fetal fibroblasts transfected with a GFP construct Bovine fetal fibroblast cells were recovered form day 50 fetuses and passaged in medium D-MEM with

10% FCS. The fetal fibroblast cells were transfected with a constructs containing the CMV/eGFP gene

(plasmid pGREEN LANTERN-1, Life Technologies) . This construct contains the reporter gene Green Fluorescence Protein (GFP) from Aequorea victoria jellyfish, which codes for a naturally fluorescent protein requiring no substrate for visualization. The GFP used is "humanized" (ie., codon sequence) and mutated to contain threonine at position 65 to enhance fluorescence peaking. The advantage of using this fluorescent gene as a reporter being that it yields bright green fluorescence when living or fixed cells are illuminated with blue light and increases our sensitivity of detection. The plasmid contains the CMV immediate early enhancer/promoter upstream of the GFP gene, followed by SV40 t-intron and polyadenylation signal. NT was performed as described above.

Table 6

Fusion and development of bovine reconstructed with nuclei from bovine fetal fibroblast cells transfected with a GFP construct and starved for 4 days and transferred to metaphase stage-enucleated oocytes or cultured for 6 h after thawing and transferred to telophase stage-enucleated oocytes

Number Fused Blastocyst

Telophase II(%) 187 131(71%) 15(11%)

Metaphase II(%) 209 169(81%) 23(14%) Table 7

Post-implantation development of cloned blastocyts derived from GFP-positive fetal fibroblasts (Table 6)

No No Non- 60 d 200 d liveborn Embryos Recipients returned positive positive

Telophase II(%) 11 6 2 1 1 1 Metaphase II(%) 15 5 4 4 3

While the invention has been described in con- nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing a reconstructed oocyte by transferring cell or nucleus from germinal or somatic cells into an enucleated host oocyte, which comprises the steps of: a) activating said host oocyte by artificial or natural means; and b) enucleating said activated host oocyte when said activated oocyte is undergoing the expulsion of a second polarbody or when said activated oocyte has recently expelled second polarbody (Tel-II); c) transferring nucleus from germinal or somatic cells into said enucleated host oocyte of step b) to obtain a reconstructed oocyte.

2. The method according to claim 1, wherein said transferred cell or nucleus is at nuclear stage GO, Gl, S, G2, or M.

3. The method of claim 1, wherein said germinal or somatic cells of step c) are cultured prior to nucleus transfer.

4. The method of claim 1, wherein said oocyte of step a) is a secondary oocyte (M-II) and said artificial means is physical or chemical means.

5. The method of claim 4, wherein said chemical means is ethanol or ionomycin.

6. The method of claim 4, wherein said physical means is selected from the group consisting of electrical means, thermal means, and irradiation technology.

7. The method of claim 1, wherein step b) is performed after oocytes are cultured for a period of time sufficient to allow for at least partial extrusion of a second polarbody.

8. The method of claim 1, wherein step b) is performed with oocytes in a medium with cytosqueletal inhibitors .

9. The method of claim 7, wherein step b) is effected by microsurgically removing said second polarbody with a portion of the cytoplasm containing chromosomes surrounding said at least partially extruded second polarbody.

10. A method of reconstituting a non-human embryo, which comprises the steps of: a) activating oocyte by artificial or natural means; b) enucleating said activated oocyte when said activated oocyte is undergoing the expulsion of a second polarbody or when said activated oocyte has recently expelled second polarbody (Tel-II) ; c) transferring a diploid nucleus or a cell in said enucleated oocyte to obtain a reconstructed oocyte with a diploid chromosomal content; and d) culturing in vi tro said reconstructed oocyte and/or transferring said reconstructed oocyte into a reproductive tract of a suitable surrogate mother to enable development into a non-human embryo.

11. The method according to claim 10, wherein said transferred cell or nucleus is at nuclear stage GO, Gl, S, G2, or M.

12. The method of claim 10, wherein said oocyte of step a) is a secondary oocyte (M-II) and said artificial means is physical or chemical means.

13. The method of claim 12, wherein said chemical means is ethanol or ionomycin.

14. The method of claim 12, wherein said physical means is selected from the group consisting of electrical means, thermal means, and irradiation technology.

15. The method of claim 13, wherein step b) is performed after oocytes are cultured for a period of time sufficient to allow for at least partial extrusion of a second polarbody.

16. The method of claim 15, wherein step b) is performed with oocytes in a medium with cytosqueletal inhibitors.

17. The method of claim 15, wherein step b) is effected by microsurgically removing said second polarbody with a portion of the cytoplasm containing chromosomes surrounding said at least partially extruded second polarbody.

18. The method of claim 17, wherein step c) is effected by introducing a single cell containing a diploid nucleus into said enucleated oocyte by cell fusion or by microinjection.

19. The method of claim 10, wherein said non-human embryo develops into a non-human animal.

20. A method for production of a transgenic non- human embryo, which comprises the steps of: a) activating oocyte by artificial or natural means; b) enucleating said activated oocyte when said activated oocyte is undergoing the expulsion of a second polarbody or when said activated oocyte has recently expelled second polarbody (Tel-II); c) transferring a transgenic diploid nucleus extracted from a cell transfected with a desired DNA construct in said enucleated oocyte to obtain a reconstructed oocyte with a diploid chromosomal content; and d) culturing in vi tro said reconstructed oocyte and/or transferring said reconstructed oocyte into a reproductive tract of a suitable surrogate mother to enable development into a non-human embryo.

21. The method according to claim 20, wherein said transferred cell or nucleus is at nuclear stage GO, Gl, S, G2, or M.

22. The method according to claim 20, which further comprises developing said non-human embryo into a fetus .

23. The method according to claim 22, which further comprises developing said fetus into an offspring.

24. The method of claim 20, wherein said non-human embryo develops into a non-human animal.

25. A transgenic embryo obtained according to the method of claim 20.

26. A transgenic fetus obtained according to the method of claim 21.

27. A transgenic offspring according to the method of claim 22.

28. A method of cloning a non-human animal by cell or nuclear transfer which comprises the steps of : a) activating oocyte by artificial means; b) enucleating said activated oocyte when said activated oocyte is undergoing the expulsion of a second polarbody or when said activated oocyte has recently expelled second polarbody (Tel-II); c) transferring a diploid nucleus or a cell in said enucleated oocyte to obtain a reconstructed oocyte with a diploid chromosomal content; and d) culturing in vi tro said reconstructed oocyte and/or transferring said reconstructed oocyte into a reproductive tract of a suitable surrogate mother to enable development into a non-human embryo.

29. The method according to claim 28, wherein said transferred cell or nucleus is at nuclear stage GO, Gl, S, G2, or M.

30. The method of claim 28, wherein said oocyte of step a) is a secondary oocyte (M-II) and said artificial means is physical or chemical means.

31. The method of claim 30, wherein said chemical means is ethanol or ionomycin.

32. The method of claim 30, wherein said physical means is selected from the group consisting of electrical means, thermal means, and irradiation technology.

33. The method of claim 28, wherein step b) is performed after oocytes are cultured for a period of time sufficient to allow for at least partial extrusion of a second polarbody.

34. The method of claim 30, wherein step b) is performed with oocytes in a medium with cytosqueletal inhibitors .

35. The method of claim 31, wherein step b) is effected by microsurgically removing said second polarbody with a portion of the cytoplasm containing chromosomes surrounding said at least partially extruded second polarbody.

36. The method of claim 32, wherein step c) is effected by introducing a single cell containing a diploid nucleus into said enucleated oocyte by cell fusion or by microinjection.

37. The method of claim 28, wherein said nucleus or cell of step c) is transgenic or non-transgenic.

38. The method of claim 28, wherein said non-human embryo develops into a non-human animal.