KR20020008398A - Telophase enucleated oocytes for nuclear transfer - Google Patents

Abstract

The present invention relates to an improved method for producing nucleus-deleted host oocytes for transplanting nuclei of embryonic cells, germ cells and somatic cells to breed or clone mammals, and to methods for producing animal embryos.

Description

Telophase enucleated oocytes for nuclear transfer

Nuclear transplantation techniques have been used extensively to propagate embryos by implanting the nuclei of early embryos into oocytes that lack cell nuclei. This technology has enabled the yield of embryos produced from parents of high genetic value to be increased, and to accelerate the annual genetic gain in animal communities. Nuclear transplantation also involves transplanting the nucleus of cell lines derived from embryonic tissue (Campbell et al., 1996, Nature 380: 64-66), fetal and adult tissues (Wilmert et al., 1997, Nature 385: 810-813). Has been used. By using the nucleus without limitation of source, nuclear transfer from cell lines has enabled the production of large numbers of genetically identical progeny as well as changes in the genetic properties of in vitro cells at all stages of progeny production, The production of transgenic mammals was made possible. In addition, proliferation (cloning) of animals with known phenotypes is possible by using cells derived from adult animals for nuclear transfer, either directly or through ex vivo procedures.

In essence, nuclear transfer technology requires donor nuclei that provide selected genetic material and host oocytes that provide the cytoplasm. These host oocytes play a role in reproducing the nucleus to ensure development into the embryo. If you own the material of the nucleus and cytoplasm, three major steps are necessary to reproduce recombinant oocytes by nuclear transfer.

The first step is to remove the nucleus from the host oocytes and remove all the genetic material. This step is usually accomplished by microsurgery removal of the chromosome from the nucleus or pronucleus of the intermediate phase of cell division. In a second step, a donor nucleus needs to be introduced into the oocyte (nuclear transplant). This step is usually accomplished by fusing the membrane of the cell of the donor nucleus with the membrane of the host oocyte. However, nuclear transfer can also directly inject the nuclei into host oocytes through the cell membranes of oocytes. The final step is to activate the inactivated host oocytes in meiosis (activation of oocytes). This step is possible by applying physical stimulation such as temperature change or electric shock to the oocytes, or by exposing the oocytes to chemical factors such as ethanol or foreign calcium.

The order of performing each of these steps may vary depending on the situation, and such order has a significant effect on the developmental capacity of recombinant oocytes.

In mice, the removal of nuclei from oocytes was achieved by visualizing the pronucleus after fertilization and removing the pronucleus by microsurgery. In other mammals where the cytoplasmic density is difficult to visualize the pronucleus, this nucleation technique is not effective. Moreover, the development rate was always low when using the oocytes from which the nucleus was removed at the pronuclear stage and the blastomere at the dividing stage as the nuclear donor. Initially, sheep could improve post-nuclear developmental stages (Willardsen, S. 1986, Nature 320: 63-65), and later in other animals by using host oocytes inactivated during the fusion phase. The developmental stage after nuclear transfer could be improved. However, in most mammals, the chromatin of the cleavage stage is not easily seen under a microscope. Wildrasen (Willardsen, S. 1986, Nature 320: 63-65) suggested that during oocyte removal, the oocytes of sheep are blindly divided into hemispheres with or without the first polar body. . M-II plates after removal of DNA vital strain (bisbenzimide; hoechst) and small amounts of cytoplasm to prevent excessive loss of cytoplasm during nuclear removal The procedure was improved by using ultraviolet irradiation to check the (MII plate). The most common oocyte denuclearization process involves exposing secondary oocytes to bisbenzamide, blindly removing the cytoplasmic nuclei that surround the polar bodies, and then exposing the oocytes to UV light to confirm that the nuclei have been correctly removed. On average, this process removes about 60-80% of oocyte nuclei. Another problem with this process is that oocytes are shown to adversely affect the cytoplasm when exposed to both ultraviolet radiation and hoechst (Smith, L. 1993 J. Reprod Fert. 99:39 -44).

As noted above, better host development after nuclear transfer can be ensured when host oocytes are used than when pronuclear-free host conjugates are used. If the oocytes from which the nucleus was removed in the M-II stage are activated or aged before fusion, a better development process is ensured. The problem with using immature and inactivated oocytes is a mismatch in the cell cycle of nuclear donor cells and host cytoplasm. Secondary (M-II) oocytes in the mid-stage phase of cell division have high levels of Maturation Promoting Factor (MPF). MPF has cellular activity involved in keeping chromatin compressed without nuclear membrane. When the mid-divided-phase nucleus of the glomeruli containing the relaxed chromatin is introduced into M-II oocytes, the nuclear membrane is rapidly lost by MPF and the chromosomes are compressed early. However, this early compaction of chromosomes is considered harmful only when it occurs during the cell cycle's DNA synthesis (S-phase). This is particularly problematic when the nucleus of the glomerulus is used as a donor-nucleus, since this nucleus stays in the S-phase for most of the time in cell division. On the other hand, if the oocytes from which the nuclei are removed are activated or aged before they are fused with the nuclear donor cells, they have lower levels of MPF, so that chromosomes are not compressed too much.

With the exception of the glomerulus, most other cell types have longer intervals between the pre-S-G stage and the post-G-Stage stages (G-2 stages), so when fused with M-II oocytes In the S-phase, the chromosome is compressed too early, and the presence of high levels of MPF causes loss of the nuclear membrane, so in the case of host oocytes in the M-II stage, the donor nucleus and putative oocyte cytoplasm It is believed that interactions between factors occur more easily. Here, the putative oocyte cytoplasmic factor refers to a factor that plays a role in reproducing the chromatin of a nucleus derived from a cell in which the differentiation stage is further advanced. There have been several literature examples in which it is advantageous to introduce more differentiated donor nuclei into inactivated M-II oocytes prior to activating recombinant oocytes. In livestock, the nucleus of an embryonic cell line ensures a very high yield of blastoccurrence and pregnancy rate when fused with oocytes that had been nucleated 4 hours prior to activation. In mice, a large number of embryos made from the nucleus of cumulus cells develop in the blastocyst stage by exposing the donor nucleus to the M-II cytoplasm for about 1 to 6 hours prior to activation (Wells et al. 1999, Viol Refred 60: 996-1005). In addition, in the case of activation and fusion at the same time, fetal development or living offspring cannot be obtained. Moreover, when using differentiated cell lines, it has been reported that activated host oocytes were used simultaneously with or after introduction of the donor nucleus (Silbeli et al. 1998, Nature Biotechnol 16: 642-646; Wilmut et al. 1997, Nature 385: 810-813). Therefore, in the case of using donor nuclei of cells other than embryonic glomeruli, it is the dominant theory in the field of cloning by nuclear transfer that a certain reproduction period is required in the cytoplasm of inactivated oocytes in order to succeed.

It is highly desirable to provide an improved method for producing nucleus deleted host oocytes for embryonic, germ and somatic cell nuclear transfer for breeding or cloning mammals.

It is also highly desirable to provide an improved method for producing embryos of animals.

The present invention relates to an improved method for producing nucleus-deleted host oocytes for transplanting nuclei of embryonic cells, germ cells and somatic cells to breed or clone mammals, and to methods for producing animal embryos.

1 is an enlightening protocol of the nucleus removal technique in the late stage of primary nuclear fission for nuclear transfer.

The present invention differs from the known art in that it discloses the use of activated oocytes as receptors for nuclei derived from cells of embryonic and somatic cells.

It is an object of the present invention to provide an improved method for producing nucleus-deleted host oocytes for embryonic, germ and somatic cell nuclear transfer for breeding or cloning mammals.

It is also an object of the present invention to provide an improved method for producing embryos of animals other than humans.

The present invention provides a method for producing nucleus-depleted host oocytes for nuclear transfer of embryonic cells, germ cells or somatic cells, comprising the following steps;

(a) activating oocytes by artificial means;

(b) immediately after the activated oocytes release the second pole body (Tel-II) or the second pole body, removing the nucleus from the activated oocytes; And

(c) transplanting nuclei of embryonic, germ or somatic cells cultured prior to nuclear transfer into (b) oocytes from which the nuclei have been removed.

The germ cells or somatic cells are cultured prior to nuclear transfer.

The oocytes of step (a) have a first polar body, and the artificial means means chemical means such as ethanol or inomycin.

Step (b) may be performed after incubation for a period of time sufficient to release the second polar body.

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

In step (b), it is effective to remove the second polar body by microsurgery along with about 1/10 of the cytoplasm surrounding the second polar body.

Preferred oocytes are secondary (M-II) oocytes.

The present invention provides a method for producing an embryo of an animal other than a human comprising the following steps;

(a) activating oocytes by artificial means;

(b) immediately after the activated oocytes release the second pole body (Tel-II) or the second pole body, removing the nucleus from the activated oocytes;

(c) introducing the nucleus of a diploid into the oocyte from which the cell nucleus has been removed to prepare an oocyte having a chromosome of the diploid; And

(d) culturing the prepared oocytes in vitro and / or introducing the prepared oocytes into the uterus of a suitable surrogate mother into embryos of animals other than humans.

The present invention provides a method for producing a transgenic embryo of an animal other than a human comprising the following steps.

(a) introducing a preferred gene construct into cultured cells selected from the group consisting of embryonic cells, germ cells and somatic cells;

(b) activating oocytes by artificial means;

(c) immediately after the activated oocytes release the second pole body (Tel-II) or the second pole body, removing the nucleus from the activated oocytes;

(d) introducing the nucleus of a diploid extracted from the cell into which the gene combination of step (a) is introduced into an oocyte from which the nucleus has been removed to prepare an oocyte having a diploid chromosome; And

(f) culturing the prepared oocytes in vitro and / or implanting the prepared oocytes into the womb of a suitable surrogate mother into embryos of animals other than humans.

Embryos of animals other than humans may be generated from animals other than humans.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for producing an embryo transplanted with nuclei of embryonic cells, germ cells and somatic cells. Nuclear transplantation methods always begin with the removal of the cell nucleus from host oocytes. After removal of the nucleus, (a) activation after fusion; (b) fusion and activation simultaneously; Or (c) a fusion step after activation. (a) The method of removing, activating and fusing cell nuclei from oocytes is most often used for embryonic glomeruli, (b) to remove cell nuclei from oocytes and to perform fusion and activation simultaneously, or (c) to nucleus cells from oocytes. Removal, activation and fusion are commonly used for more differentiated donor nuclei. Although other methods have already been disclosed in nuclear transfer methods (US Pat. No. 4,994,384; US Pat. No. 5,057,420; US Pat. No. 5,843,754 and International Patent Applications PCT / GB96 / 02098, PCT / US98 / 00002, PCT / US98). / 12800, PCT / US98 / 12806 and PCT / US97 / 12919, the present invention discloses a novel sequence of steps in the nuclear transfer method (FIG. 1).

As shown in FIG. 1, step 1 involves the activation of secondary (M-II) oocytes by artificial means. Step 2 is performed immediately after the activation step when the oocytes are in the process of releasing a second pole (Tel-II) or just after release. Step 3 is optional for preparing oocytes with diploid chromosomes. Transplantation of the cell nucleus.

Stage 1 (Activation of Oocytes)

Oocytes can be obtained in vivo or ex vivo and are cultured in a maturation medium. After aging, the cumulus cells are removed from the oocytes and the oocytes with the first polar body are activated by chemical means such as ethanol or inomycin. After activation, the oocytes are cultured for several hours before releasing the second polar body.

Step 2 (remove the nucleus of oocytes)

After activation, oocytes are placed in media containing cytosqueletal inhibitors to facilitate microsurgery. Only oocytes from which the second polar body is released or partially released are used. The second polar body is removed by microscopic surgery to remove approximately 1/10 of the cytoplasm surrounding the approximately second polar body.

Stage 3 (nuclear transplant)

After removal of the nucleus, one cell with the diploid nucleus is introduced into the oocytes from which the nucleus is removed by cell fusion or microinjection (nuclear transplantation). The oocytes thus prepared are implanted into the uterus of the surrogate mother immediately or without undergoing in vitro culture and continue to develop.

The invention is illustrated with reference to the following examples. However, the following examples do not limit the scope of the present invention.

Example 1 Removal of Cell Nuclei at the End of Cell Division

A follicle with a diameter of 2 to 8 mm was extracted from the ovaries of the meat shop cattle. Oocytes with homogeneous cytoplasm and multiple cell stacks were selected and aged for 1 hour after follicle cell extraction. After 28 hours of aging, a pile of cells were removed from the oocytes and oocytes with the first polar body were used for the experiment. Oocytes were exposed to 7% ethanol for 5 minutes, washed and placed in another cycle of aging medium. One hour before microscopic surgery, oocytes were placed in cytochalasin B and placed in a suitable location for micromanipulation. In the oocytes immediately after release or immediately after release, the second pole was removed along with about 10% of the cytoplasm surrounding it. After microscopic surgery, oocytes were fixed with 10% formalin and stained with 5 μg Hoechst 33342 and observed by UV epi-fluorescence. Oocytes without chromatin were judged to be successful cell nuclei. When finely manipulated to the time tested in Table 1, removal of the nucleus was successful in most oocytes. Since the cell nucleus removal technology has high efficiency, it is not necessary to identify oocytes by DNA staining and ultraviolet light. Twenty-four hours after the initiation of in vitro maturation, the efficiency of cell nucleation was lowered when blindly removed about 30% of the cytoplasm surrounding the first polar part of oocytes in the medium phase (59%).

Removal of nuclei at the end of successful cell division at different times following exposure to stimuli to activate secondary oocytes into unipotent germ cells. Time after activation 3 hours 4 hours 5 hours Total time Manipulation of successful enucleation (%) 37 38 43 118 36 37 40 113 (97%) (97%) (93) (96%)

Example 2 Nuclear Transplantation of Morphogenetic blastomeres

Bovine secondary oocytes were aged in vitro and the nuclei were removed using the technique described above (elimination of nuclei at the end of cell division). Embryos of the morula phase were digested and each blastomeres inserted into the perivitelline of the nucleus-deleted follicle cells. The membranes of the blastomeres and the oocytes were fused by applying an electric shock that fuses the donor membrane and the receptor cell membrane. The electrical parameter used is 60 μsec double pulse of 1.5 kvolts per cm. After fusion, the embryos were incubated for 7 days in Menezo's B2 medium with fetal bovine serum.

Fusion and Development of Bovine Oocytes Recombinant to the Nucleus of the Glomerulus of the Loss Phase Recovered for 5 Days After IVF Number fusion Blastocyst Number of nuclei Terminal II (%) 215 129 (58%) 49 (38%) 126 ± 11 Medium term II (%) 248 151 (60%) 24 (16%) 84 ± 9

Example 3 Nuclear Transplantation of Non-Deficient Bovine ES Cells

Bovine embryonic liver (ES) cells were obtained from 8 day old embryos of the blastocyst stage, all generated ex vivo. ICMs are shown on fibroblasts of mitomycin-inactivated mice. The resulting embryonic liver (ES) cell line was isolated by brief exposure to trypsin using a narrow pipette.

The isolated cells were introduced into the perivitelline of the nucleus-deleted oocytes and subjected to an electric shock that fuses the donor membrane with the membrane of the receptor cell. The electrical parameters used are 100 μsec double pulses of 1.5 kvolts per cm. To obtain better fusion results, an electric shock was applied immediately after introducing the nucleus of the donor cell into the perivitelline. After fusion, the embryos were cultured for 7 days in Menezo's B2 medium with fetal bovine serum.

Fusion and Development of Recombinant Bovine Oocytes into the Nucleus of Embryonic Liver (ES) Cells Exposed to FCS 5% Number fusion decomposition Blastocyst Terminal II (%) 38 11 (30%) 5 (45%) 3 (27%) Medium term II (%) 33 12 (36%) 2 (17%) 1 (8%)

Example 4 Nuclear Transplantation of ES Cells of Serum-Deficient Bovine

Prior to fine manipulation, bovine embryonic liver (ES) cells were cultured in batches of 0.5% FCS for 5 days. As described above, embryonic liver (ES) cells were isolated, introduced into the perivitelline of nucleus-deleted oocytes, and subjected to electrical shock to fuse the donor membrane with the membrane of the receptor cell. After fusion, the embryos were incubated for 7 days in Menezo's B2 medium with fetal bovine serum.

Fusion and development of bovine oocytes recombined into the nucleus of (deficient) bovine embryonic liver (ES) cells exposed to low concentrations of 0.5% of FCS Number fusion decomposition Blastocyst Terminal II (%) 38 13 (34%) 3 (23%) 2 (27%) Medium term II (%) 42 13 (31%) 4 (31%) 1 (15%)

Example 5 Nuclear Transplantation of Deficient or Non-Deficient Fetal Fibroblasts

Cells from fetal fibroblasts were recovered from 50 day old fetuses and transferred to D-MEM medium containing 10% FSC. Non-deficient fibroblasts were harvested after incubation for 2 days. Serum-deficient cells were exposed for 5 days to a batch containing 0.5% serum prior to nuclear transfer (NT). Nuclear transplantation was performed as described above.

Fusion and development of oocytes recombined into the nucleus of fetal fibroblasts exposed to 5% FCS for 20 hours (deficient) after 5 days exposure (deficient) to seeding at 0.5% low concentration of FCS. Deficient serum Nondeficient Serum Number Convergence Blast Number Convergence Blast Terminal II (%) 69 52 (75%) 2 (4%) 105 67 (64%) 9 (13%) Medium term II (%) 60 39 (65%) 9 (24%) 114 92 (81%) 12 (13%)

Example 6 Nuclear Transplantation of Deficient and Non-deficient Fetal Fibroblasts Infected with GFP Combination

Fetal fibroblasts were recovered from 50 day old fetuses and placed in D-MEM medium containing 10% FSC. Fetal fibroblasts were infected with the CMV / eGFP gene (plasmid pGREEN LANTERN-1, biotechnology) combination. The combination includes a reporter gene, the green fluorescent protein (GFP) gene of Aequorea victoria jellyfish, which encodes a natural fluorescent protein that requires any substrate for visualization. Do not The GFP used is humanized (eg, coding sequence) and contains a threonine residue mutated at 65 residues that augment the fluorescence peak. The advantage of using the fluorescent gene as a reporter gene is that live cells or fixed cells are colored in blue, which can increase detection sensitivity. The plasmid contains a CMV early enhancer / promoter upstream of the GFP gene, an SV40 t-intron downstream and a polyadenonylation signal sequence. Nuclear transplantation (NT) was performed as described above.

Fusion and development of cows infected with the GFP combination and deficient for 4 days, transplanted or dissolved in the nuclear cell deficient follicles of the mid-cell division, and cultured for 6 hours, followed by recombination into the nucleus of the fetal fibroblasts transplanted into the late cell division-nuclear deficient follicle cells. Number Convergence Blastocyst Terminal II (%) 187 131 (71%) 15 (11%) Medium term II (%) 209 169 (81%) 23 (14%)

Post-transplantation development of proliferated blastocysts derived from GFP-positive fetal fibroblasts (Table 6) Radish embryo Receptor-free Non-conversion 60d positive 200d positive liveborn Terminal II (%) 11 6 2 One One One Medium term II (%) 15 5 4 4 3

Although the present invention has been disclosed by the above specific embodiments, the scope of the present invention is not limited to the above embodiments, and those skilled in the art to which the present invention pertains various modifications and variations within the scope of the present invention described in the claims. Such modifications and variations are also included in the scope of the present invention.

Claims (37)

Method for producing recombinant oocytes by transplanting nuclei or cells of germ cells or somatic cells into cell nucleus-deleting host oocytes comprising the following steps (a) activating host oocytes by artificial or natural means; (b) immediately after the activated oocytes release the second pole body (Tel-II) or the second pole body, removing the nucleus from the activated oocytes; And (c) introducing a nucleus of a germ cell or a somatic cell into the oocyte from which the nucleus is removed, thereby obtaining a recombinant oocyte. The method of claim 1, wherein the nucleus or cells to be transplanted is characterized in that the cell division step is G0, G1, S, G2 or M. The method of claim 1, wherein the germ cells or somatic cells of step (c) are cultured prior to nuclear transfer. The method of claim 1, wherein the oocytes of step (a) are secondary (M-II) oocytes and the artificial means are physical or chemical means. 5. The method of claim 4 wherein the chemical means is ethanol or inomycin. 7. The method of claim 6, wherein the physical means is selected from the group consisting of electrical mean, thermal mean and radiation mean. The method of claim 1, wherein step (b) is performed after the oocytes have been cultured for a time sufficient to release at least partially the second polar body. The method of claim 1, wherein step (b) is performed with oocytes in a medium containing a cytosqueletal inhibitor. 8. The method of claim 7, wherein step (b) is performed by microsurgically removing the second polar body with a portion of the cytoplasm containing at least partially released chromosomes around the second polar body. How to feature. A method for producing an embryo of an animal other than a human, comprising the following steps. (a) activating oocytes by artificial or natural means; (b) immediately after the activated oocytes release the second pole body (Tel-II) or the second pole body, removing the nucleus from the activated oocytes; And (c) grafting the nucleus or cells of the diploid to the oocytes from which the nucleus has been removed to produce recombinant oocytes containing the chromosomes of the diploid; And (d) culturing the recombinant oocytes ex vivo and / or implanting the recombinant oocytes into the uterus of a suitable surrogate mother to develop into embryos of animals other than humans. 11. The method of claim 10, wherein said nucleus or cell to be transplanted is characterized in that the cell division step is G0, G1, S, G2 or M. The method of claim 10, wherein the oocytes of step (a) are secondary (M-II) oocytes and the artificial means are physical or chemical means. 13. The method of claim 12, wherein the chemical means is ethanol or inomycin. 13. The method of claim 12, wherein the physical means is selected from the group consisting of electrical mean, thermal mean, and radiation mean. 14. The method of claim 13, wherein step (b) is performed after the oocytes have been cultured for a time sufficient to release at least partially the second polar body. 16. The method of claim 15, wherein step (b) is performed with oocytes in a medium containing a cytosqueletal inhibitor. 16. The method of claim 15, wherein performing step (b) by microsurgically removing the second pole body with a portion of the cytoplasm containing at least partially released chromosomes around the second pole body. How to feature. 18. The method according to claim 17, wherein step (c) is performed by introducing one cell containing a diploid nucleus into an oocyte from which said nucleus has been removed by cell fusion or microinjection. The method of claim 10, wherein the embryos of animals other than humans live in animals other than humans. A method for producing a transgenic embryo of an animal other than a human, comprising the following steps. (a) activating oocytes by artificial or natural means; (b) immediately after the activated oocytes release the second pole body (Tel-II) or the second pole body, removing the nucleus from the activated oocytes; And (c) grafting the gene transfer diploid nucleus extracted from the cells into which the desired DNA construct has been introduced into the oocytes from which the nucleus has been removed to prepare recombinant oocytes containing the chromosomes of the diploid; And (d) culturing the recombinant oocytes ex vivo and / or implanting the recombinant oocytes into the uterus of a suitable surrogate mother to develop into embryos of animals other than humans. 21. The method of claim 20, wherein the nucleus or cell to be transplanted has a cell division step of G0, G1, S, G2 or M. The method of claim 20, further comprising the step of developing an embryo of said non-human animal into a fetus. The method of claim 22, further comprising the step of developing said fetus as offspring. 21. The method of claim 20, wherein embryos of animals other than humans are generated from animals other than humans. A transgenic embryo produced by the method of claim 20. A transgenic embryo obtained by the method of claim 21. A transgenic embryo obtained by the method of claim 22. A method of cloning an animal other than a human by cell or nuclear transfer, comprising the following steps. (a) activating oocytes by artificial means; (b) immediately after the activated oocytes release the second pole body (Tel-II) or the second pole body, removing the nucleus from the activated oocytes; And (c) grafting the nucleus or cells of the diploid to the oocytes from which the nucleus has been removed to produce recombinant oocytes containing the chromosomes of the diploid; And (d) culturing the recombinant oocytes ex vivo and / or implanting the recombinant oocytes into the uterus of a suitable surrogate mother to develop into embryos of animals other than humans. 29. The method of claim 28, wherein the nucleus or cell to be transplanted has a cell division step of G0, G1, S, G2 or M. 29. The method of claim 28, wherein the oocytes of step (a) are secondary (M-II) oocytes and the artificial means are physical or chemical means. 31. The method of claim 30, wherein said chemical means is ethanol or inomycin. 31. The method of claim 30, wherein the physical means is selected from the group consisting of electrical mean, thermal mean and radiation mean. 29. The method of claim 28, wherein step (b) is performed after the oocytes have been cultured for a time sufficient to release at least partially the second polar body. 31. The method of claim 30, wherein step (b) is performed with oocytes in a medium containing a cytosqueletal inhibitor. 32. The method of claim 31, wherein performing step (b) by microsurgically removing the second polar body with a portion of the cytoplasm containing at least partially released chromosomes around the second polar body. How to feature. 33. The method of claim 32, wherein step (c) is performed by introducing one cell containing a diploid nucleus into an oocyte from which said nucleus has been removed by cell fusion or microinjection. 29. The method of claim 28, wherein the nucleus or cell of step (c) is with or without a gene.