RU2216591C2 - Method for obtaining human cloned embryos due to applying the method of interspecific nuclear transplantation - Google Patents

Abstract

FIELD: medicine, biology. SUBSTANCE: the present method deals with the following stages: obtaining the lines of human donor somatic cells; maturation of oocytes taken out of cow's ovary, in vitro; removal of oocyte- surrounding cumulus cells, dissection of the part of sparkling area of maturated oocytes by obtaining a fissure to press out the part of cytoplasm through this fissure including the first polar body at obtaining enucleated recipient oocytes; transportation of nucleus into recipient oocyte due to injection of donor cells into nucleus-free recipient oocytes followed by successive electrofusion and activation of electrofused cells to obtain embryos; postactivation and cultivation of embryos in vitro. Obtained cloned human embryos could be applied to obtain human embryonic stem cells to be widely used both in medicine and biology. EFFECT: higher efficiency. 12 cl, 4 dwg, 5 ex, 11 tbl

Description

Field of Invention
This invention relates to a method for producing human cloned embryos by applying a method of interspecific transplantation of cell nuclei, and more particularly, to a method for producing human cloned embryos by using a method of interspecific transplantation of cell nuclei, through which somatic cell nuclei derived from human tissue are transferred to mature oocytes originating from a cow. The invention also relates to cloned human embryos obtained by the method described above.

BACKGROUND OF THE INVENTION
For a long time, it was believed that the production of animals should be carried out through fertilization, which includes male and female gametes. However, tremendous efforts have been made to obtain cloned animals with identical species and identical genetic characteristics.

 Recently, various recombinant plants with desirable traits of valuable cultivated plants have been successfully obtained thanks to developments in biotechnology and genetic engineering (see: Schweizer et al., Plant Journal, 20: 541-552, 1999). As for animals, there have been many successful examples of the production of cloned animals, which include, for example, cloned lambs (see: Wilmut et al., Nature, 385: 810-813, 1997), cloned cows (see: Wells et al ., Reprod. Fertil. And Develop., 10: 369-378, 1998) and cloned mice (see: Wakayama et al., Nature, 394: 369-374, 1998). Since obtaining cloned animals cannot be realized without high technology created on the basis of biotechnology, an assessment of technological development in the relevant areas was considered the norm.

 Meanwhile, it was known that animal stem cells have the ability to develop into any organ, which prompted the study of the mechanism of their differentiation into each organ by their production and cultivation. When conducting this study, it is important to use materials with identical tissue specificity to reduce variation among many different studies. However, it is obvious that suitable materials with identical tissue specificity are not available all the time. Although the somatic cell cloning method has recently facilitated the production of materials with identical tissue specificity, it has been less satisfactory for human tissue.

 In these circumstances, there were compelling reasons for developing a method for producing human embryonic stem cells with identical tissue specificity.

SUMMARY OF THE INVENTION
According to this invention, it was found that human embryonic stem cells can be successfully obtained using a method of interspecific transplantation of cell nuclei, which includes the fusion of cow oocytes and human skin cells and culturing human cloned embryos in vitro to the morul / blastocyst stage.

 The first objective of this invention is, therefore, providing a method for producing cloned human embryos using a method of interspecific nuclear transplantation.

 Another objective of this invention is the provision of cloned human embryos obtained in this way.

BRIEF DESCRIPTION OF THE DRAWINGS
The above and other objectives and features of the present invention will become apparent from the following description, given together with the accompanying drawings, in which:
Figure 1 is a photograph of somatic donor cells.

 Figure 2 is a photograph showing the process of cutting a zona pellucida (transparent zone, i.e., shiny egg shell) of a recipient oocyte with a holding pipette and a cutting pipette.

 Figure 3 is a photograph showing the process of enucleation by removing the first polar body and nucleus from the recipient oocyte.

 Figure 4 is a photograph showing the process of transferring a somatic cell into an enucleated (non-nuclear) oocyte with a holding pipette and an injection pipette.

DETAILED DESCRIPTION OF THE INVENTION
A method for producing cloned human embryos of the present invention comprises the steps of obtaining lines of somatic donor cells collected from a human; maturation of oocytes collected from the ovary of a cow, in vitro; removal of cumulus cells (an egg mound on the wall of the Graaf bubble where the egg is placed) surrounding oocytes, cutting part of the shiny area of mature oocytes and squeezing part of the cytoplasm, which includes the first polar body, to obtain enucleated recipient oocytes; transferring the nucleus to the recipient oocyte by injection of donor cells into enucleated oocytes, followed by successive electro-fusion and activation of electro-fused cells to produce embryos; post-activation and cultivation of embryos in vitro.

 The method for producing cloned embryos of the present invention is further illustrated as follows.

Stage 1: Obtaining donor cells
Somatic cell lines collected from humans are prepared as donor cells: although cells collected from humans are not a limitation of donor cells, preferred cell lines include skin cells or fibroblasts collected from the umbilical cord of newborns. A more preferred cell line for donor cells are skin cells isolated from skin tissue. These cell lines are obtained using a well-known known method (see: Mather & Barnes, Methods in Cell Biology, vol. 57, Animal Cell Culture Methods, Academic Press, 1998) with some modifications.

For example, cells or fibroblasts of the skin of the umbilical cord of the newborn are washed and ground. Then, these cells are subjected to trypsin and type II collagenase treatment under conditions of 39 ^° C, 5% CO ₂ , followed by cultivation in DMEM (Dulbecco's modified Eagle medium) supplemented with non-essential amino acids, 10% FCS (fetal calf serum) and 1% penicillin-streptomycin mixture (10,000 U / ml penicillin, 10 mg / ml streptomycin) under the conditions described above.

 These somatic cell lines are stored using subculture, a culture of serum starvation or freezing. Subculture of donor cell lines is carried out at regular intervals by replacing the old medium with a new medium after trypsinization. Cultivation with serum starvation is carried out using DMEM supplemented with 0.5% FCS and the method of Wilmut et al. (see: Wilmut et al., Nature, 385: 810-813, 1997). Cell lines thus stored are used for a later stage as donor cells.

Stage 2: Receiving recipient oocytes
Immature oocytes collected from bovine ovary are brought to maturation in vitro: immature oocytes are taken from the ovary in TCM199 washing medium containing 10 mM HEPES (N- [hydroxyethyl] piperazine-N '- [2-ethanesulfonic acid]) and matured culturing these cells in a TCM199 culture medium (containing Na-pyruvate, penicillin-streptomycin) supplemented with estradiol, FSH (follicle-stimulating hormone) and FCS for 16-22 hours at 39 ^° C, 5% CO ₂ .

Stage 3: Enucleation of recipient oocytes
After removing the cumulus cells (egg mound on the wall of the Graaf bubble) surrounding mature recipient oocytes and cutting a part of the oocyte brilliant zone, part of the cytoplasm including the first polar body is removed from the oocytes to obtain enucleated oocytes: first, the cumulus cells surrounding mature oocytes are removed physically denuding pipette in the washing medium TCM199 containing hyaluronidase. Then, denudated oocytes are washed with TCM199 washing medium and transferred to a solution of cytochalazine B. To enucleate denudated oocytes, part of the brilliant zone of denudated oocytes is pierced by a cutting pipette with the formation of a gap through which 10-15% of the cytoplasm, including the first polar body, can be squeezed out of the oocytes. Enucleated oocytes are washed and incubated in TCM199 culture medium. The indicated cytochalazine B solution is prepared by diluting cytochalazine B dissolved in DMSO (dimethyl sulfoxide) with TCM199 culture medium.

Stage 4: Electro-fusion of donor cells with recipient oocytes and activation of electro-fused cells
Donor cells are transferred to recipient oocytes, followed by electrofusion and activation of electrofused cells: before donor cells are injected into recipient oocytes, enucleated oocytes are washed with TCM199 culture medium and transferred to a PHA-P (phytohemagglutinin) solution. Then, donor cells are transferred to enucleated oocytes by injection of donor cells into a gap made in the shiny area of oocytes in a PHA-P solution.

Electrofusion is performed using an Electro Cell Manipulator (BTX ECM2001). Reconstructed embryos in a mannitol solution supplemented with a washing solution TCM199 are placed in a chamber with two electrodes, one on each side. Before embryos are placed with their donor cells facing the cathode in the chamber, this chamber is filled with a mannitol solution. After electrofusion of embryos by applying an alternating current pulse of 0.75-2.00 kV / cm twice with an interval of one second for 15 μs each time, electrofused embryos are washed with mannitol solution and TCM199 washing medium, incubated in a cytochalazine B solution and activated. Electrofusion and activation are carried out simultaneously, provided that the electrofusion is carried out in an environment with mannitol containing Ca ²⁺ . Otherwise, activation is carried out after electrofusion. When conducting electrofusion in a Ca ^2+-free medium with mannitol, the activation stage is carried out by incubating the embryos in a solution of ionomycin in the dark. Then, ionomycin is removed from the embryos by washing them with TCM199 washing medium containing FCS or BSA. The specified solution of ionomycin is obtained by diluting ionomycin dissolved in DMSO, washing medium TSM199 containing BSA.

Stage 5: Post-activation and cultivation of in vitro embryos
Embryos are post-activated and cultured in vitro: activated embryos incubated in TMS199 washing medium containing FCS or BSA are post-activated by incubation in a solution of cycloheximide or DAMP (4-dimethylaminopurine) and cultivated in vitro under 5% CO ₂ or a mixture of 5% CO ₂ , 7% O ₂ and 88% N ₂ . Said cycloheximide solution or DAMP solution is prepared by adding cycloheximide dissolved in ethanol or DAMP to in vitro culture media, respectively. In vitro culture media include mTALP media (see table 1) (tables 1-11 see the end of the description), mSOF (see table 2) and mCR2aa (see table 3), all of which contain NaCl, KCl, NaHCO ₃ , NaH ₂ PO ₄ , CaCl ₂ , Na-lactate, glucose, phenol red, BSA, kanamycin, essential amino acids that are not essential amino acids and L-glutamine.

Optionally, in vitro cultured embryos are stored by freezing for later use and thawed when intended to be used. To freeze embryos, they are washed with PBS containing FCS, placed in a freezing medium containing penicillin-streptomycin, CaCl ₂ , glucose, MgCl ₂ , Na-pyruvate and PBS. Then the embryos in the medium for freezing are subjected to slow freezing, followed by rapid freezing in liquid N ₂ . When removing frozen embryos from liquid N ₂ and thawing, they are placed in the air for approximately 5 seconds and then thawed in warm water. To remove the medium for freezing from thawed embryos, they are placed sequentially in environments containing glycerin from its high concentration to low concentration.

 Based on the above method, the inventors obtained an embryo, SNU6 (human somatic cell line), using human skin cells as nuclear donors. This embryo was deposited by the International Depository, CCTS, and the Korean Collection of Type Cultures (Korean Collection for Type Cultures; KRIBB # 52, Oundong, Yusong-ku, Taejon, 305-333, Republic of Korea) on June 19, 2000 under accession number CCTC 0805BP.

 The invention is further illustrated in the following examples, which should not be construed as limiting the scope of the invention.

Example 1: Obtaining donor cells and recipient oocytes
To obtain donor cells, tissue collected from human skin was washed with PBS (buffered phosphate saline, GIBCO BRL, Life Technologies, USA) and ground to a size of 100 mesh. Then this tissue was incubated in PBS containing 0.25% trypsin, 1 mM EDTA and 1 mg / ml type II collagenase for 1 hour at 39 ^° C, 5% CO ₂ . After cleavage of the tissue with enzymes, it was centrifuged at 1500 rpm for 2 minutes and suspended in DMEM (Dulbecco's modified Eagle GIBCO BRL medium, Life Technologies, USA) supplemented with 10% FCS, 1% NEAA (non-essential amino acids) and 1% penicillin-streptomycin mixture. The suspension was transferred to a cell culture dish and incubated at 39 ^° C, 5% CO ₂ to give a somatic cell line. After that, the cells were trypsinized in a solution containing 0.25% trypsin and 1 mM EDTA, and the number of cells was adjusted to 2 x 10 ⁴ cells / ml to place aliquots of cells in Eppendorf tubes.

 Figure 1 depicts somatic cells isolated as separate cells for a nuclear donor.

 On the other hand, for recipient oocytes, follicles that were 2-6 mm in diameter were aspirated from Korean cows' ovaries with a 10 ml syringe having an 18G needle. Then this follicular fluid was transferred to a 100 mm dish with a grid (the length between the lines was 1 cm) drawn on its bottom, and oocytes with a homogeneous cytoplasm and a sufficient number of layers of cumulus cells around them were screened. The selected oocytes were washed three times with 2 ml of TCM199 washing medium (see table 4) in 35 mm plates and then once with TCM199 culture medium (see table 5). Finally, oocytes were cultured in TCM199 culture medium containing 0.1% estradiol solution (see table 6), 2.5% follicle-stimulating hormone solution (see table 7) and 10% FCS, to obtain recipient oocytes.

Example 2: Somatic Cell Nuclear Transfer
The recipient oocytes obtained in Example 1 were washed once with TCM199 washing medium and transferred to a 0.1% hyaluronidase solution (Sigma Chemical Co., USA) prepared by mixing 1 ml of TCM199 washing medium with 111 μl of the initial hyaluronidase solution (10 mg / ml in the washing medium TCM199). After removal of cumulus cells from oocytes in the presence of a 0.1% hyaluronidase solution, denudated oocytes were washed three times and incubated in TCM199 washing medium. Then, these oocytes were transferred to a solution of cytochalazine B (Sigma Chemical Co., USA) prepared by mixing 1 ml of TCM199 washing medium containing 10% FCS with 1 μl of a cytochalazine stock solution (7.5 mg / ml in DMSO) and a brilliant zone each oocyte was cut using a micromanipulator to form a gap through which 10-15% of the cytoplasm can be squeezed out of the oocyte to produce an enucleated (coreless) oocyte. The enucleation step is more specifically illustrated as follows: the working cup was placed on the micromanipulator cup and the micromanipulator was provided with a holding pipette on its left shoulder and a cutting pipette on its right shoulder. Then, the holding pipette and the cutting pipette were placed in the direction of 9 hours and 3 hours, respectively, and adjusted for free movement in all directions by placing the controller (regulator) of the pipettes in the middle. These two pipettes were further adjusted to prevent them from touching the work cup, and their tips were placed in the middle of the micro droplet by moving them up and down the micro droplet. Then, the oocytes were transferred from the TCM199 washing medium into a cytochalazine B solution using washing pipettes with a bell (inner diameter> 200 μm). The micromanipulator was first focused on the oocyte using its coarse adjustment handle and fine adjustment handle, and this focus was further adjusted by moving these two pipettes up and down. The oocyte was placed with its first polar body oriented in the direction of 12 hours and a retention pipette was placed near this oocyte in the direction of 9 hours of the oocyte to fix the oocyte by applying hydraulic pressure. Figure 2 shows the process of cutting a shiny area of an oocyte using a holding pipette and a cutting pipette. As shown in figure 2, the oocyte was pierced by a cutting pipette (2) from the direction of 1 hour to the direction of 11 hours with extreme care so as not to damage the cytoplasm of the oocyte. After that, hydraulic pressure was applied to the retention pipette (1) to separate the oocyte (3) and the retention pipette was brought into contact with the cutting pipette penetrating through the bordering glossy zone on the upper part of the first polar body to cut part of the shiny zone by touching these two pipettes . The oocyte gap made as described above was used both for enucleation and for injection of a donor cell. Figure 3 shows an enucleation process that removes the first polar body and nucleus from an oocyte. As shown in figure 3, the oocyte (3) was placed so that its gap was oriented vertically, held with a holding pipette (1) on its lower part to prevent it from moving, and gently pressed on its upper part with a cutting pipette (2) to obtain enucleated oocyte. The enucleated oocyte was washed three times with TCM199 washing medium and incubated in TCM199 culture medium.

 After this, donor cells obtained in advance were transferred to enucleated oocytes using a micromanipulator. First, a 4 μl injection droplet was placed in the middle of the working cup using a PHA-P solution prepared by mixing 400 μl of a washing solution of TCM199 and 100 μl of a stock solution of PHA-P (phytohemagglutinin) (0.5 mg / ml in a washing solution of TCM199). Then two micro droplets were made for donor cells, one of which was placed higher and the other below the injection micro droplets on the same working cup using PBS containing 1% FCS. After distributing these microdroplets with mineral oil, the working cup was placed on the micromanipulator cup.

 A cutting pipette mounted on a micromanipulator was replaced with an injection pipette. Enucleated oocytes were washed three times with TCM199 washing medium and transferred to an injection microdroplet. Donor cells were pulled into an injection pipette and transferred to an injection micro-droplet. Figure 4 shows the process of transferring a somatic cell into an enucleated oocyte. As shown in FIG. 4, an enucleated oocyte was placed with its slit oriented in the direction of 1 hour, fixed with a holding pipette and injected with a donor cell through a slot using an injection pipette and hydraulic pressure to obtain a reconstructed embryo. This embryo was washed three times with TCM199 washing medium and incubated in TCM199 washing medium.

Example 3: Electrofusion and Activation
The reconstructed embryos were electrofused using the Electrocell Manipulator cell manipulator (ECM 2001, BTX, USA), followed by activation. 15 μl of a mannitol solution containing 0.28 M mannitol, 0.5 mM HEPES (pH 7.2), 0.1 mM MgSO ₄ and 0.05% BSA was added to the TCM199 culture medium containing reconstructed embryos using a pipette with a bell for washing. After a 1-minute incubation in this medium, the embryos were incubated for 1 minute in a mannitol solution supplemented with a washing solution TCM199, and finally transferred to a mannitol solution using a pipette with a flushing socket. The chamber (chamber 3.2 mm 453) of the cell electric manipulator was filled with a mannitol solution supplemented with TCM199 washing medium, and then the embryos were placed in this chamber so that their part with the donor cell was facing the cathode. After electrofusion of the embryos by applying an alternating current pulse of 0.75-2.00 kV / cm twice with an interval of 1 second for 15 μs each time, they were transferred to the TCM199 washing medium and washed three times with this medium from mannitol solution.

 To activate electrofused embryos, they were incubated in the dark for 4 minutes in a solution of ionomycin (Sigma Chemical Co., USA), which was a TCM199 washing medium containing 5 μM ionomycin and 1% BSA. A stock solution of ionomycin was prepared by dissolving 1 mg of ionomycin in 1.34 ml of DMSO. Activated embryos were incubated for 5 minutes in a 35 mm dish containing TCM199 washing medium supplemented with 10% FCS to remove ionomycin from these embryos.

Example 4: Post-activation and in vitro cultivation of electrofused embryos
Activated embryos were postactivated for 4 hours in 25 μl of a cycloheximide solution (Sigma Chemical Co., USA) prepared by adding a stock solution of cycloheximide (10 mg / ml in ethanol) to an in vitro culture medium, mTALP, at a final concentration of 10 μg / ml . Then the embryos were screened and the selected embryos were incubated for 7 days under conditions of 39 ^o C, 5% CO ₂ . During cultivation, embryo development was monitored as time passed (see table 8).

 As shown in table 8, it was clearly demonstrated that the method of interspecific nuclear transplantation allows to obtain the development of cloned human embryos to the morula / blastocyst stage, which ultimately facilitates the production of human embryonic stem cells from developed morula / blastocysts.

 Based on the above method, the inventors obtained an embryo, SNU6 (human somatic cell line), using human skin cells as nuclear donors. This embryo was deposited by the international depository, CCTS (Korea Type Culture Collection; KRIBB # 52, Oundong, Yusong-ku, Taejon, 305-333, Republic of Korea) on June 19, 2000 under accession number CCTC 0805BP.

Example 5: Freezing and thawing of embryos and transplantation
Embryos were frozen for long-term storage. First, the freezing medium (see tables 9 and 10) was distributed in 35 mm plates and the freezer was turned on to maintain the temperature at -5 ^° C. The embryos selected for freezing were washed with PBS containing 10% FCS and incubated in freezing medium in for 20 minutes. The embryos were then pulled into a 0.25 ml French straw so that the straw had a freezing medium containing embryos in the middle and two layers of air at both ends. After sealing by heating the straws using heated tweezers, it was placed in the freezer, kept at -5 ^o C for 5 minutes and separated with tweezers, previously cooled with liquid N ₂ . After separation, the straw was cooled at a speed of -0.3 ^o C / min to -30 ^o C, kept for 10 minutes when the temperature reached -30 ^o C. Finally, these embryos were stored in a tank with liquid N ₂ .

To thaw frozen embryos, a thawing medium containing PBS supplemented with 20% FCS was prepared in 35 mm plates and supplemented with glycerol to obtain thawing media, each of which contained 0%, 3%, and 6% glycerol (see tables 9 and 11 ) Then the frozen straw was removed from the liquid N ₂ , kept in air for 5 seconds and thawed in a tank (> 20 cm in diameter) containing warm water (30 ^o C). After thawing, the straw was cut in places of air layers at both ends and the medium containing the embryos was collected. Embryos were examined under a microscope. To remove the freezing medium from the embryos, they were successively incubated in thawing media containing 6% glycerol, 3% glycerol and 0% glycerol, each 5 minutes.

 As clearly shown and explained above, the present invention provides a method for producing cloned human embryos using an interspecific nuclear transplantation method, comprising transferring nuclei derived from human somatic cells to oocytes derived from a cow and human cloned embryos obtained by this method. According to the method of the present invention, the production of cloned human embryos can be used to obtain human embryonic stem cells, which can be widely used for various purposes, such as the treatment of diseases and other applications in pharmaceuticals and medical science.

 A variety of modifications of the invention, other than those shown and described herein, will be apparent to those skilled in the art from the previous description. It is understood that such modifications are within the scope of the appended claims.

Claims (13)

 1. A method for producing cloned human embryos, comprising the steps of: (i) obtaining lines of somatic donor cells collected from a human; (ii) maturation of oocytes collected from cow ovary in vitro; (iii) removing the cumulus cells surrounding the oocytes, cutting a part of the brilliant zone of mature oocytes to obtain a gap and squeezing part of the cytoplasm, including the first polar body, through this gap to obtain enucleated (nuclear-free) recipient oocytes; (iv) transferring the nucleus to the recipient oocyte by injection of donor cells into enucleated (non-nuclear) recipient oocytes, followed by successive electro-fusion and activation of electro-fused cells to produce embryos; (v) post-activation and cultivation of embryos in vitro.  2. The method for producing cloned human embryos according to claim 1, wherein the somatic cell lines obtained in stage (i) include human skin cells or fibroblasts collected from the umbilical cord of newborns.  3. The method for producing cloned human embryos according to claim 1, wherein the somatic cell lines are stored using subculture (reseeding) of the culture with serum starvation or freezing.  4. The method for producing cloned human embryos according to claim 1, wherein the cumulus cells surrounding the oocytes in stage (iii) are physically removed with a denuding pipette after treatment with a hyaluronidase.  5. The method for producing cloned human embryos according to claim 1, wherein the enucleation of oocytes in stage (iii) is carried out by forming a gap on the oocyte by cutting it with a micromanipulator; placing this oocyte with its slit oriented vertically and holding the bottom of the oocyte with a holding pipette to prevent cell movement; squeezing the upper part of the oocyte with a cutting pipette to release 10-15% of the cytoplasm containing the first polar body from the oocyte through this gap.  6. The method for producing cloned human embryos according to claim 1, wherein the transfer of the nucleus in stage (iv) is carried out by injection of a donor cell into a recipient enucleated (nuclear-free) oocyte through a gap made in the shiny area of the oocyte.  7. The method for producing cloned human embryos according to claim 1, wherein the electrofusion in step (iv) is carried out by applying an alternating current pulse of 0.75-2.00 kV / cm twice with an interval of 1 s for 15 μs each time. 8. The method for producing cloned human embryos according to claim 1, wherein activation in step (iv) occurs simultaneously with electrofusion, provided that the electrofusion is performed in a medium containing Ca ²⁺ . 9. The method for producing cloned human embryos according to claim 1, wherein the activation in step (iv) is carried out in a solution of ionomycin in the dark, provided that the electrofusion is performed in a Ca ²⁺ free medium.  10. The method for producing cloned human embryos according to claim 1, wherein the postactivation in step (v) is carried out by culturing the embryos in a cycloheximide solution or a DMAP (4-dimethylaminopurine) solution.  11. The method for producing cloned human embryos according to claim 1, wherein the in vitro cultivation in step (v) is carried out by culturing the postactivated embryos in mTALP, mSOF or mCR2aa medium. 12. The method for producing cloned human embryos according to claim 1, further comprising the step of storing the embryos cultured in vitro in step (v) for later use after freezing the embryos in freezing medium containing penicillin-streptomycin, CaCl ₂ , glucose, MgCl ₂ , Na-pyruvate and phosphate buffered saline.  13. A method for producing cloned human embryos according to claim 1 for producing an SNU6 embryo (human somatic cell line, CCC 0805BP), using human skin cells and Korean cow oocytes as donors of nuclei and recipient oocytes, respectively. Priority on points and signs:
06/30/1999 PP 1-13;
01/04/2000 established by the amendments to the claims.

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