RU2205537C2 - Method for obtaining cloned tiger due to applying method of interspecific nuclear transplantation - Google Patents

Abstract

FIELD: gene engineering. SUBSTANCE: the present innovation deals with methods for animal cloning. The present method to obtain cloned tigers deals with obtaining the lines of tiger's donor somatic cells, isolation of oocytes out of either cow's or female cat's ovaries and their maturation in vitro. Then cumulus cells and surrounding oocytes should be removed to obtain enucleated recipient oocytes. Then one should transfer nucleus of donor cells into recipient oocyte with subsequent electrofusion and activation of electrofused cells that leads to obtain an embryo. Then obtained embryos should be postactivated and cultured in vitro. Slightly grown embryos should be transferred into mother surrogate followed by obtaining cloned tigers. The present innovation is applied for keeping population of tigers and in saving other species being under the threat of dying off. EFFECT: higher efficiency. 12 cl, 4 dwg, 12 tbl

Description

Field of Invention
This invention relates to a method for producing cloned tigers by applying a method of interspecific transplantation of cell nuclei, and more particularly, to a method for producing cloned tigers by using a method of interspecific transplantation of cell nuclei by which somatic cell nuclei obtained from tiger tissue are transferred to mature oocytes originating from a cow or cats. The invention also relates to cloned tiger embryos and cloned tigers 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.

 Cloning of zygotes was known to be possible only in the case of amphibians for 30 years, until success was achieved in obtaining cloned offspring by replacing the pronucleus of a unicellular zygote in mice (see McGrath and Solter, Science, 220: 1300-1302, 1983). Despite this first success in animal cloning, a similar success in industrial animals (see Wakayama et al., Nature, 394: 369-374, 1998) was reported much later since obtaining cloned mice using mature zygote oocytes and blastomeres after 2 The cell stage had some problems, such as a decrease in reprogramming.

 Regarding the production of cloned industrial animals using nuclear transfer, the first report was that the offspring were obtained from a sheep by using 8-16-cell zygote blastomeres as donor cells (see Wiladsen, Nature, 320: 63-65, 1986) . Since then, it was believed that only zygote blastomeres with totipotency, due to which a cell can be differentiated into any single cell, can be cloned by nuclear transfer. However, the first cloned sheep was obtained by introducing nuclei from somatic cells (see Wilmut et al, Nature, 385: 810-813, 1997), which amended the previous development theory and allowed to report many successful examples in obtaining cloned cows (see Wells et al., Reprod. Fertil. and Develop., 10: 369-378, 1998) and pigs.

 Embryos obtained using somatic cells of existing wild animals, and the method of obtaining these embryos were perceived as very valuable for the permanent conservation of the genetic traits of rare or endangered species. However, there have not yet been reports of successful cloning of existing wildlife. In approaches to cloning these wild animals, cloning has encountered difficulties in obtaining recipient oocytes in the case of cloning animals that are rare or protected. Thus, their cloned animals must be obtained by using interspecific transplantation of cell nuclei and oocytes originating from closely related species. This method of interspecific nuclear transplantation was published earlier (see Dominko et al., Biol. Reprod., 60 (6): 1496-1502 (1999). However, it was aimed at the use of oocytes and somatic cells of industrial animals, which already had a lot of research work was done, which made the application of this method in obtaining cloned wild animals difficult.

 In these circumstances, there are compelling reasons for the research and development of an improved method for producing cloned wild animals through the use of interspecies transplantation of nuclei and somatic cells of wild animals.

 SUMMARY OF THE INVENTION

 According to the present invention, it was found that cloned tiger embryos and cloned tigers developed from these embryos can be obtained using a method of interspecific transplantation of cell nuclei, including the fusion of oocytes from a cow or cat with somatic tiger cells.

 Thus, the first objective of this invention is to provide a method for producing cloned tigers using a method of interspecific nuclear transplantation.

 Another objective of this invention is the representation of cloned tiger embryos obtained in this way.

 Another objective is to present cloned tigers derived from somatic cells obtained using the method.

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 tigers of the present invention comprises the steps of: obtaining somatic donor cell lines collected from a tiger; maturation of oocytes collected from the ovary of a cow or cat 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 in vitro embryos; and transfer of cultured embryos to “surrogate” (replacement) mothers to produce cloned tigers.

 A method for producing cloned tigers of the present invention is further illustrated as follows.

Stage 1: Obtaining donor cells
Somatic cell lines collected from a tiger are obtained as donor cells, although cells collected from a tiger are not a limitation on donor cells, preferred cell lines include cells collected from a uterine lavage fluid, endometrium, oviduct, ear or muscle, cumulus cells or fetal fibroblasts, which are obtained using a generally known method (see Mather & Barnes, Methods in Cell Biology, vol. 57, Animal Cell Culture Methods, Academic Press, 1998) with some modifications.

For example, cells are harvested by adding PBS (phosphate buffered saline) containing 1% penicillin-streptomycin (Gibco, 10,000 U / ml penicillin, 10 mg / ml streptomycin) to the uterine lavage fluid, followed by centrifugation. Cells collected from uterine lavage fluid are cultured in DMEM (Dulbecco's modified Eagle's 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) in conditions of 39 ^o C, 5% CO ₂ .

 Uterine epithelial cells collected from the endometrium or oviduct are washed with the indicated PBS, trypsinized and cultured under the conditions described above.

For cumulus cells, the cumulus-oocyte complexes are treated with a hyaluronidase solution to isolate the cumulus cells surrounding oocytes. Cumulus cells are trypsinized for 30-60 minutes under 39 ^o C, 5% CO ₂ prior to culturing with the method described above.

As for the fibroblasts of the ears and fetal fibroblasts, they are obtained from the inner side of the skin lined with cartilaginous tissue, and from tissue collected from the trunk and extremities of the fetus, respectively, by aseptic washing and grinding of these tissues, followed by treatment with trypsin and type II collagenase under conditions of 39 ^o C 5% CO ₂ . These cells are also cultured similarly to the cultivation of somatic donor cells described above.

 These somatic cell lines are stored using subculture (passivated culture), serum starvation culture, or freezing. Subculturing (reseeding) 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 a cow or cat’s ovary are brought to maturation in vitro: immature oocytes are taken from the ovary in TCM199 flushing medium containing 10 mM HEPES (N- [hydroxyethyl] piperazine N '- [2-ethanesulfonic acid]) and adjusted before maturation in various culture media, depending on the type of animal from which the oocytes were obtained. For maturation of oocytes obtained from a cow, TCM199-1 culture medium is used (see table 1). For maturation of oocytes collected from cats, cells are cultured in TCM199-2 culture medium (see table 2) supplemented with human chorionic gonadotropin (hCG). In both cases, the cultivation was carried out for 16-22 hours under conditions of 39 ^o C, 5% CO ₂ .

Stage 3: Enucleation of recipient oocytes
After removal of the cumulus cells (egg mound on the wall of the Graaf bubble) surrounding mature recipient oocytes and cutting of 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, cumulus cells surrounding mature oocytes, removed physically with a denuding pipette in a TCM199 washing medium 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 the embryos by applying a direct current pulse of 0.75-2.00 kV / cm twice with an interval of one second for 15 μs each time, the electrofused embryos are washed with a 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 (see table 3), mSOF (see table 4) and mCR2aa (see table 5), all of which contain NaCI, KC1, NaHCO ₃ , NaH ₂ PO ₄ , CaCl ₂ , Na-lactate, glucose, phenol red, BSA, kanamycin, essential amino acids, non-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.

Stage 6: Obtaining Cloned Tigers
In vitro cultured embryos are transferred to “surrogate” mothers to obtain tigers: embryos in PBS containing FCS are implanted into the uterus of “surrogate” mothers.

 Based on the above method, the inventors obtained an embryo, SNU5 (Korean tiger embryo), using tiger ear cells and Korean cow oocytes as donors of nuclei and recipient oocytes, respectively. 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 March 10, 2000 under accession number CCTC 0752BP.

 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, ethanol and betadine were disinfected around the apical ear tissue of an adult male tiger after shaving the hair near this tissue. Skin tissue (1-2 cm ² by area) was collected with sterilized instruments, transferred to 50 ml test tubes containing phosphate buffered saline (PBS, GIBCO BRL, Life Technologies, USA) with 0.5% penicillin (10000 U / ml) - streptomycin (10 mg / ml). Then, the cartilage tissue and the hair-containing skin were separated from said collected skin tissue by sterilized scissors and surgical blades to obtain tissue on the inside of the skin lined with cartilage tissue for donor cells. This fabric was washed with PBS and crushed 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's medium, GIBCO BRL, Life Technologies, USA) supplemented with 10% FCS, 1% NEAA (essential amino acids) and 1 % penicillin-streptomycin mixture. The suspension was transferred to plates for culturing cells and incubated at 39 ^° C, 5% CO ₂ to obtain 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 • 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 6) in 35 mm plates and then once with TCM199-1 culture medium (see table 1). Finally, oocytes were cultured in TCM199 culture medium containing 0.1% estradiol solution (see table 7), 2.5% follicle-stimulating hormone solution (see table 8) 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) obtained 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, denuded 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) obtained 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 special care in order 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 a 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, an injection microdrop of 4 μl was placed in the middle of the working cup using a PHA-P solution obtained by mixing 400 μl of a washing solution of TCM199 and 100 μl of an initial 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 above and the other below the injection micro droplets on the same working plate 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 an Electro 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 embryos by applying a direct 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 washing medium TCM199 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 (see table 3), in 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 .

 Based on the above method, the inventors obtained an embryo, SNU5 (Korean Tiger NT Embryo), using tiger ear cells and Korean cow oocytes as donors of nuclei and recipient oocytes, respectively. This embryo was deposited by the International Depository, CCTS, and the Korean Type Culture Collection (Korean Collection for Type Cultures; KRIBB # 52, Oun-dong, Yusong-ku, Taejon, 305-333, Republic of Korea) on March 10, 2000, under the accession number of CCTS 0752BP.

Example 5: Production of Embryos by Using Oocytes Derived from a Cat
The inventors of the present invention obtained and cultured embryos by applying the method described in Examples 1-4, except that oocytes obtained from a cat were used as recipient oocytes.

Example 6: Freezing and thawing of embryos and transplantation
The embryos obtained in examples 4 and 5 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. Then the embryos were pulled into a 0.25 ml French straw in such a way that the straw had a freezing medium containing embryos in the middle and two layers of air. After sealing by heating, the straws at both ends using heated tweezers were placed in a freezer, kept at -5 ^° 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.

 Thawed embryos were placed in PBS containing 20% FCS and pulled into a straw. Then they were transferred to the uterus of the "surrogate" mother.

Example 7: Comparison of embryos using different donor cells
To compare the differences between the embryos obtained using different recipient oocytes, the embryos obtained in examples 4 and 5 were implanted in “surrogate” mothers and compared in relation to the following indicators: the number of electrofused oocytes, the degree of electrofusion (%), the degree of division (%) , the number (%) of 8-cell embryos, the number (%) of 16-cell embryos, the number (%) of 32-cell embryos, the number (%) of developed morula / blastocysts, the number of transferred embryos and the number of pregnancies after embryo transfer (see table 12). The number (%) of morula / blastocyst represents the ratio of embryos that have developed in vitro culture to the stage immediately before implantation, to the total number of embryos obtained by nuclear transfer.

 As shown in table 12, the use of cow oocytes showed a higher degree of development and the possibility of pregnancy than the use of cat oocytes. This can be explained by the fact that many previous studies on the use of cow oocytes helped establish optimal conditions for nuclear transplantation, while large studies have not been conducted on the use of cat oocytes.

 Previous studies have shown that industrial animals, which have been extensively studied, can be used for interspecific transplantation of cell nuclei. However, none of these studies have been successful in obtaining cloned offspring. Thus, in connection with the circumstances described above, this invention has made great progress in obtaining cloned animals derived from a somatic cell, since it has obtained cloned offspring derived from a somatic cell.

 As clearly shown and explained above, the present invention provides a method for producing cloned tigers using a method of interspecific nuclear transplantation, comprising fusing somatic tiger cells with oocytes obtained from a cow or cat. It also provides cloned tiger embryos and cloned tigers developed from these embryos obtained in this way. In accordance with the method of this invention, the genetic traits of rare or endangered species can be permanently preserved by using interspecific nuclear transplantation to obtain their cloned embryos as a way of preserving wild animals. It is expected that, in addition to the conservation of wild animals, the method of the present invention can be applied to the development of many related applications, including a method of interspecific nuclear transplantation.

 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. Then

Claims (13)

 1. A method for producing a cloned tiger, comprising the steps of: (i) obtaining lines of donor somatic cells of a tiger; (ii) in vitro maturation of ovum from the ovary of a cow or cat; (iii) removing the cumulus cells surrounding the ovum, cutting a portion of the shiny area of the mature egg cell to produce a gap and squeezing a portion of the cytoplasm including the first polar body through this gap to produce an enucleated recipient egg cell; (iv) transferring the nucleus to the recipient egg by introducing the whole donated somatic cell into the enucleated recipient egg, followed by successive electro-fusion and activation of the electro-fused cell to produce an embryo; (v) post-activation and cultivation of the embryo in vitro; and (vi) transferring the cultured embryo to a surrogate mother to produce a cloned tiger.  2. The method according to claim 1, characterized in that the somatic cell lines obtained in stage (i) include cells from uterine lavage, endometrium, oviduct, ear or muscle, cumulus cells or fetal fibroblasts.  3. The method according to claim 1, characterized in that the somatic cell lines are stored using subculture (reseeding), serum-free culture or freezing.  4. The method according to claim 1, characterized in that the cumulus cells surrounding the eggs in stage (iii) are physically removed with a denuding pipette after treatment with a hyaluronidase.  5. The method according to claim 1, characterized in that the enucleation of the egg in stage (iii) is carried out by the formation of a gap in the egg by cutting it with a micromanipulator; placing this egg with a slit oriented vertically and holding the bottom of the egg with a holding pipette to prevent cell movement; squeezing the upper part of the egg with a cutting pipette to release 10-15% of the cytoplasm containing the first polar body from the egg through this gap.  6. The method according to claim 1, characterized in that the nuclear transfer in stage (iv) is carried out by introducing a donor cell into a recipient enucleated egg cell through a slit made in the shiny area of the egg cell.  7. The method according to claim 1, characterized in that the electrofusion in step (iv) is carried out by applying two direct current pulses of 0.75-2.00 kV / cm with an interval of 1 s, each 15 μs long. 8. The method according to claim 1, characterized in that the activation in stage (iv) occurs simultaneously with the electrofusion, provided that the electrofusion is performed in a medium containing Ca ²⁺ . 9. The method according to claim 1, characterized in that 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 according to p. 1, characterized in that the post-activation in stage (v) is carried out by culturing the embryo in a solution of cycloheximide or a DMAP solution (4-dimethylaminopurine).  11. The method according to p. 1, characterized in that the in vitro cultivation in stage (v) is carried out by culturing the postactivated embryo in mTALP, mSOF or mCR2aa medium. 12. The method according to claim 1, characterized in that it further comprises the step of storing the embryo cultured in vitro in step (v) for later use after freezing the embryo in a freezing medium containing penicillin-streptomycin, CaCl ₂ , glucose, MgCl ₂ , sodium pyruvate and phosphate buffered saline.  13. The method according to claim 1, characterized in that it provides for the production of an Korean tiger embryo NT, CCC 0752BP, (SNU5) using a tiger ear cell and a Korean cow egg (Bos taurus coreanae), respectively, as a nucleus and recipient egg donor. Priorities for items:
06/30/1999 according to claims 1-3-12;
01/28/2000 according to claims 2 and 13.

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