JP2003503045A - Clonal tiger production method using xenogeneic nuclear transfer technology - Google Patents

Abstract

(57) Abstract The present invention provides a method for producing clone tigers by the technique of xenogeneic nuclear transfer. In the method for producing a cloned tiger according to the present invention, a somatic cell obtained from a tiger is cultured to prepare a donor somatic cell line; immature oocytes are collected, cultured, and matured from bovine or feline ovaries. Removing the cumulus cells surrounding the oocyte, cutting a part of the zona pellucida of the mature oocyte to make a cut, and removing a part of the cytoplasm including the first polar body from the cut. Thereby producing an enucleated recipient oocyte; injecting said donor cell into said enucleated recipient oocyte to implant a nucleus into said recipient oocyte; Performing fusion and activating the electrofused cells to obtain embryos; post-activating the activated embryos in vitro and then in vitro culturing; transplanting the in vitro cultured embryos into surrogate mother animals By the clone tiger Comprising; step of childbirth. The invention has broad applicability to the permanent storage of the genetic traits of rare or endangered wild animals in the form of embryos.

Description

Detailed Description of the Invention

[0001] BACKGROUND Field of the Invention The present invention relates to a method of production related to clone the tiger using a cross-species nuclear transfer technology. More specifically, the present invention provides a method for producing a cloned tiger using the xenogeneic nuclear transfer technique of introducing a somatic cell nucleus derived from a tiger tissue into an oocyte derived from a bovine or feline, and a method produced by the method. Clone tiger embryo and clone tiger.

[0002] Conventionally , it has been recognized that an organism such as an animal is capable of normal ontogeny only by connecting male and female gametes, but an individual having the same appearance and genetic trait is artificially generated. In order to produce it, the hard work has been done.

Cloning of fertilized eggs has succeeded in producing clonal progeny by replacing the pronucleus of mouse 1-cell stage embryos (McGrath and Solter, Science, 22).
(See 0: 1300-1302, 1983) For almost 30 years, it was known to be possible only for amphibians. However, despite the initial success of cloning animals, the production of cloned mice using mature oocytes and blastomeres from the 2-cell stage onwards has several consequences, including reduced reprogramming. The success in commercial animals was reported much later because of the problem of (1) (Wakayama et al., Nature,
394: 369-374, 1998).

Regarding the production of cloned commercial animals by the technique of nuclear transfer, Wila
dsen) obtained the first offspring of sheep using the embryonic blastomeres of the 8-16 cell stage of sheep as donor cells (see Wiladsen, Nature, 320: 63-65, 1986), and has since differentiated into whole cells. Only the blastomeres with the totipotency that
It has been recognized as a cell that can be cloned by nuclear transfer. However, the introduction of somatic cell-derived nuclei creates the first cloned sheep (Wi-
See lmut et al., Nature, 385: 810-813, 1997), a modification of the conventional embryological theory. It should be noted that the technology was rapidly developed and supplemented within just two to three years, and cows (Wells et al., Reprod. Fertil. And Develop., 10: 3
69-378, 1998) and successful production of cloned animals such as pigs.

Cloned embryos produced using existing wildlife somatic cells and methods of producing the embryos are of great importance because they can permanently preserve the genetic traits of rare or endangered wildlife. It is evaluated as a new technology. However, successful cloning of existing wild animals has not yet been reported. In such wild animal cloning, if the target animal is rare or is a protected animal, it is difficult to obtain the recipient oocyte. It can be used to produce cloned animals by cross-species nuclear transfer. Although technology for such heterogeneous nuclear transfer has been previously announced (Dominko
et al., Biol. Reprod., 60 (6): 1496-1502, 1999), which uses oocytes and somatic cells of commercial animals that have already been studied. There were problems with practical application in the production of cloned wild animals.

[0006] Therefore, there has been a strong demand for the development of xenogeneic nuclear transfer technology and technology for producing cloned wild animals by using somatic cells of wild animals.

[0007] According to the Summary of the Invention invention, by using a cross-species nuclear transplantation technique fusing oocytes from bovine or feline tiger-derived somatic cells, clones tiger generated from cloned tiger embryos and embryo Was able to produce.

Therefore, a main object of the present invention is to provide a method for producing cloned tiger by the xenogeneic nuclear transfer technique.

[0009] Another object of the present invention is to provide a cloned tiger embryo produced by the above method.

Another object of the present invention is to provide a somatic cell-derived cloned tiger produced by the above method.

[0011] The method of producing clones tiger The present invention includes the steps of preparing a donor somatic cell lines were cultured somatic cells obtained from tigers; collecting and culturing immature oocytes from bovine or feline ovary And maturation; after removing the cumulus cells surrounding the oocyte, a part of the zona pellucida of the mature oocyte is incised to make a cut, and the cytoplasm containing the first polar body from the cut To remove enucleated recipient oocytes by injecting the donor cells into the enucleated recipient oocytes to transfer nuclei to the recipient oocytes. And then performing electrofusion to activate the electrofused cells to obtain an embryo; post-activating the activated embryo in vitro and then in vitro culturing; Transplanting into mother animals More, step of giving birth to clone tigers; comprising.

Hereinafter, the method for producing a cloned tiger of the present invention will be described in more detail by dividing it into stages.

Step 1 : Preparation of Donor Cells Somatic cell lines obtained from tiger are prepared as donor cells. At this time, the cells of the tiger are not particularly limited, but cells isolated from the uterine perfusate of the tiger, the endometrium, the fallopian tube, the ear or the muscle, the cumulus cells or the fetal fibroblasts are preferable. These are Ma (Ma
ther) and Barnes (Mather & Barnes, Methods in Cell Biolo
gy, Vol. 57, Animal Cell Culture Methods, Academic Press, 1998) to produce a cell line.

For example, phosphate buffered saline (PBS) containing P / S antibiotics (Gibco; penicillin 10000 U / ml, streptomycin 10 mg / ml) was added to the uterine perfusate, centrifuged and washed, and then 10%. DMEM (Dulbecco's modified Eagles medium) supplemented with fetal bovine serum (FBS), non-essential amino acids and 1% P / S antibiotics
) At 39 ° C. and 5% CO ₂ .

Uterine epithelial cells are collected from a part of the endometrium or oviduct and washed with the above PBS. Then, after trypsin treatment, the cells are cultured under the above conditions.

For cumulus cells, the cumulus cell-oocyte complex is treated with a hyaluronidase solution to separate the cumulus cells surrounding the oocyte and the cumulus cells at 39 ° C., 5% C
After treatment with trypsin for 30 to 60 minutes under O ₂ , the cells are cultured under the above conditions.

[0017] Ear fibroblasts and fetal fibroblasts were collected from the endothelium in contact with cartilage tissue and the torso and extremities of the fetus, and the tissue was aseptically washed and chopped, followed by trypsin and II. Type collagenase is added and treated under the conditions of 39 ° C. and 5% CO ₂ , and then cultured under the above conditions.

Somatic cell lines are preserved by subculture, serum starvation culture or freezing. Subculture of the donor cell line is performed at regular intervals, replacing the old medium with a new one after trypsinization. Serum starvation culture is performed by DMEM supplemented with 0.5% FBS and the method of Wilmat et al. (Wilmut et al., Nature, 385: 810-813).
, 1997). Also, the cell line preserved by the above method is used as a donor cell in the subsequent step.

Second stage : Preparation of recipient oocytes Immature oocytes are collected and cultured and matured from bovine or feline ovaries. 10 mM HEPES (N- [hydroxyethyl] piperazine-N ′-[2-
Ethane sulfonic acid]) containing TCM199 washing medium (Tissue Culture Medium
In 199), immature oocytes are selected from the ovaries, and then the medium is changed according to the kind of the origin animal of the oocytes to be matured. In this case, the medium used depends on the animal that provided the oocyte. To mature bovine-derived oocytes, TCM199-
In order to mature the cat-derived oocytes by culturing in 1 medium (see Table 1), the cells are cultured in TCM199-2 medium (see Table 2) containing human chorionic gonadotropin (hCG).

[0020]

[Table 1] TCM199-1 medium

[0021]

[Table 2] TCM199-2 medium

Step 3 : Enucleation of recipient oocyte After removing the cumulus cells of the mature recipient oocyte, a part of the zona pellucida of the oocyte is incised to remove the first polar body. An enucleated oocyte is prepared by removing a part of the containing cytoplasm. First, mature recipient oocytes are placed in a TCM199 washing medium in which hyaluronidase is dissolved, the cumulus cells are physically removed by an exposure pipette, and then washed with TCM199 washing medium. Then, the oocytes from which the cumulus cells have been removed are transferred to the cytochalasin B solution, and the zona pellucida of the oocyte is incised with a dissection pipette to make a cut, and then the whole cytoplasm of the oocyte is cut from the cut. 1
Enucleation is carried out by removing the cytoplasm containing the first polar body in an amount corresponding to 0% to 15%. Next, the enucleated oocyte is washed with TCM199 washing medium and cultured. At this time, the cytochalasin B solution used was prepared by adding cytochalasin B to DMS.
A solution dissolved in O (dimethyl sulfoxide) was diluted with TCM199 medium.

Step 4 : Electrofusion of donor cells and recipient oocytes and activation of electrofused cells The donor cells are transplanted to enucleated oocytes, followed by electrofusion and electrofusion. Activate the fused cells. First, before injecting donor cells into enucleated oocytes, the enucleated oocytes were washed with TCM199 medium and transferred to a PHA-P (phytohemagglutinin) solution, and then the donor cells were PHA- The oocytes in P solution are injected and transplanted into the zonula above the zona pellucida.

Electrofusion is performed by Electro Cell Manipulator (BTX ECM2001). First, the reconstructed embryo is placed in a mannitol solution containing TCM199 washing medium, which is placed in a chamber having two electrodes (one on each side). The chamber is filled with the mannitol solution and then the embryos are placed with the donor cells facing the cathode. Next, the voltage is 0.75 kV / cm to 2.00 kV / cm and the time is 15 μm.
s, the embryo is electrofused by passing a direct current under the condition that the number of times is 2 times at 1 second intervals. The fused embryo is washed with a mannitol solution and a TCM199 washing medium, and then incubated in the cytochalasin B solution to be activated. However, when electrofusion is performed in a mannitol solution containing Ca ²⁺ , electrofusion and activation occur simultaneously, but in other cases, activation is performed after electrofusion. When electrofusion is carried out in Ca ²⁺ -free mannitol solution, the activation step is carried out by incubating the embryos in ionomycin solution in the dark. Then, ionomycin is removed from the embryo by washing with TCM199 washing medium supplemented with fetal bovine serum or BSA. The ionomycin solution was prepared by dissolving ionomycin in DMSO and diluting it with TCM199 washing medium containing BSA.

[0025]Stage 5 : Post activation of embryo and in vitro culture   After the activated embryo is post-activated, it is cultured in vitro. That is, fetal bovine serum
Or activation incubated in TCM199 wash medium supplemented with BSA
Cycloemimide solution or DAMP (4-dimethylaminopurine)
After incubation in solution and post-activation, 5% CO_TwoOr 5% CO_Two,
7% O_TwoAnd 88% N_TwoIn vitro culture under the condition of the mixture. Above cyclo
The ximide solution is a medium for in vitro culture of cycloheximide dissolved in ethanol.
The DAMP solution was prepared by adding DAMP to the medium for in vitro culture.
Prepare. The medium for in vitro culture includes NaCl, KCl, NaHCO 3. _Three , NaH_TwoPO_Four, CaCl_Two, Sodium lactate, glucose, phenol red,
Contains BSA, kanamycin, essential amino acids, non-essential amino acids, and glutamine
MTALP medium (see Table 3), mSOF medium (see Table 4), mCR2aa medium
(See Table 5).

In some cases, an in vitro-cultured embryo can be cryopreserved and then thawed before use when necessary. In the case of freezing embryos, first, the fertilized eggs to be frozen are washed with PBS containing fetal bovine serum to obtain P / S antibiotics, CaCl ₂ , glucose, Mg.
Place in a freezing solution containing Cl ₂ , Na-pyruvate and PBS and apply the slow freezing method (slow f
Reezing) and then liquid nitrogen. When thawing the frozen embryo in this manner, the embryo taken out from liquid nitrogen is placed in the air for about 5 seconds and then put in warm water to thaw it. The thawed embryos are sequentially placed in a medium containing high to low concentrations of glycerol and incubated to remove the frozen liquid in the embryos.

[0027]

[Table 3] mTALP medium

[0028]

[Table 4] mSOF medium

[0029]

[Table 5] mCR2aa medium

Step 6 : Delivery of cloned tiger The above-mentioned in vitro-cultured embryo is transplanted into a surrogate mother to give birth to a tiger. That is, embryos in PBS containing fetal bovine serum are transplanted into the uterus of a surrogate mother.

The present inventors have produced a cloned tiger embryo by using the above method, using a tiger ear cell as a nuclear donor and a Korean bovine oocyte as a recipient.
KCTC (Korean Coll), an international depository institution located at 52 Uogakudo Cave, Yuseong-gu, Daejeon, Korea, on March 10, 2000.
Section for Type Cultures; KRIBB # 52) with deposit number KCTC0752BP.

Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the technical scope of these embodiments.

Example 1 Preparation of Donor Cells and Recipient Oocytes First, in order to prepare donor cells, the hair of the tip of the ear of a male tiger (adult) was shaved and disinfected with ethanol and betadine. Use sterilized instruments to remove skin tissue (1-2c
m ² ), 0.5% P / S antibiotic (penicillin 10,000 U / ml)
, Streptomycin 10 mg / ml) in phosphate buffered saline (PBS, G)
ibco BRL, Life Technologies, USA) was transferred to a 50 ml test tube. Then, using sterilized scissors and a surgical knife, the skin containing cartilage tissue and body hair was separated from the collected skin tissue, and the tissue of the inner skin in contact with the cartilage tissue was collected.
The collected tissue was washed with phosphate-buffered saline (PBS), cut into 100 mesh pieces, and added with 0.25% trypsin, 1 mM EDTA and 1 mg / ml type II collagenase. In phosphate buffered saline at 39 ° C, 5% C
Incubate for 1 hour under O ₂ environment. The enzyme digested tissue was then centrifuged at 1500 rpm for 2 minutes and DMEM (Dulbecco's modified Eagle) containing 10% fetal bovine serum, 1% non-essential amino acids and 1% P / S antibiotics.
's medium, Gibco BRL, Life Technologies, USA) was suspended in a medium, transferred to a cell culture dish, and cultured under the conditions of 39 ° C. and 5% CO ₂ to obtain a somatic cell line. Then, the cells were trypsinized in a solution containing 0.25% trypsin and 1 mM EDTA, the cell number was adjusted to 2 × 10 ⁴ cells / ml, and this was dispensed into an Eppendorf test tube to give donor cells. Prepared.

[0034]   Figure 1 shows somatic cells isolated as single cells of nuclear donor.

On the other hand, from a bovine ovary, a follicle having a diameter of about 2 to 6 mm was aspirated using a 10 ml syringe equipped with an 18 G injection needle, and a follicle having a diameter of about 2 to 6 mm was sucked into a 100 mm dish (lattice graduations at 1 cm intervals were provided on the bottom). After transfer, oocytes with well-adhered cumulus cells and homogeneous cytoplasm were selected. The selected oocytes were treated with TCM199 washing medium (Table 6).
(See reference) is washed 3 times with a 35 mm dish dispensed in 2 ml portions, and then TCM199-1
It was washed once with the medium (see Table 1). Finally, recipient oocytes were prepared by culturing in TCM199 medium containing 0.1% estradiol solution (see Table 7), 2.5% follicle stimulating hormone solution (see Table 8) and 10% FBS. .

[0036]

[Table 6] TCM199 washing medium

[0037]

[Table 7] Estradiol solution

[0038]

[Table 8] Follicle-stimulating hormone solution

Example 2 : Somatic cell nuclear transfer The recipient oocytes obtained in Example 1 were washed once with TCM199 washing medium, and then hyaluronidase (Sigma Chemical Co., USA) stock solution (TCM199 washing medium). 111 μl of 10 mg per 1 ml) and 1 ml of TCM199 washing medium were mixed, the oocytes were transferred to a hyaluronidase solution adjusted to a final concentration of 0.1% to remove cumulus cells, and then washed 3 times with TCM199 washing medium, Incubated. Next, cytochalasin B was added to DMSO until the concentration became 7.5 mg / ml.
(Sigma Chemical Co., USA) 1 μl of stock solution and 1 ml of TCM199 washing medium supplemented with 10% fetal bovine serum were mixed to prepare oocytes and transferred to a cytochalasin B solution, and a micromanipulator was used. After making a cut in the zona pellucida of each oocyte, the amount corresponding to 10% to 15% of the total cytoplasm was removed from the oocyte through this to enucleate the oocyte.

To describe the enucleation process more specifically, a working dish was placed on a micromanipulator plate, and a fixing pipette was attached to the left arm of the micromanipulator and an incision pipette was attached to the right arm. Thereafter, the fixing pipette was placed at 9 o'clock and the incision pipette was placed at 3 o'clock. The pipette adjuster was placed in the middle and the pipette was moved up, down, left and right. While moving the two pipettes up and down in the microdroplets, the angle was adjusted so that the pipettes did not touch the edge of the dish and the tip of the pipette was placed in the center of the microdroplets. The oocytes were transferred from the TCM199 washing medium to the cytochalasin B solution using a washing mouse pipette having an inner diameter of 200 μm or more. The coarse and fine screws of the micromanipulator were then used to first focus on the oocyte and then the two pipettes were moved up and down to further adjust the focus. The oocyte was placed with its first polar body facing the 12 o'clock direction, and a fixing pipette was placed in close contact with the oocyte at the 9 o'clock direction of the oocyte, and then the oocyte was applied by applying a fluid pressure. Fixed FIG. 2 shows a process of incising the zona pellucida of an oocyte with a fixing pipette and an incision pipette. As can be seen from FIG. 2, the zona pellucida was pierced with the incision pipette (2) from the 1 o'clock direction and then penetrated in the 11 o'clock direction while being careful not to damage the cytoplasm. After that, a fluid pressure is applied to the fixing pipette (1) to separate the oocyte (3),
After the fixing pipette was brought into contact with the transparent band at the upper end of the first polar body through which the cutting pipette passed, two transparent pipettes were rubbed to cut the transparent band. The above cuts in oocytes were used for both enucleation and injection of donor cells. FIG. 3 shows the enucleation process of removing the first polar body and nucleus of the oocyte. As can be seen from FIG. 3, the oocyte (3) is placed with its cuts oriented vertically, and the fixing pipette (1) is placed under the oocyte so that the oocyte does not move downward. After being supported, the incision pipette (2) was gently pushed from above the oocyte to enucleate. The oocytes enucleated in this manner are treated with TCM19.
9 washes 3 times with wash medium and incubated in TCM199 culture medium.

Next, donor cells were transplanted to the enucleated oocytes using a micromanipulator. First, 100 mg of a solution prepared by dissolving 5 mg of PHA-P (phytohemagglutinin) in 10 ml of TCM199 washing medium and TCM199 washing medium 4
Make 4 μl of microdroplets for injection in the middle of the working dish with PHA-P solution prepared by mixing 00 μl, and add 4 μl of microdroplets of donor cells with PBS containing 1% fetal bovine serum. I made one each on the top and bottom. After applying mineral oil to these microdroplets, the working dish was placed in the micromanipulator.

After exchanging the incision pipette attached to the micromanipulator with the injection pipette, the enucleated oocyte was washed three times with TCM199 washing medium and transferred to a microdroplet for injection. Donor cells were transferred to transplant microdroplets using an injection pipette. FIG. 4 shows the process of transplanting somatic cells into enucleated oocytes. As can be seen from Fig. 4, the cut line of the enucleated oocyte was placed at 1 o'clock direction and fixed with a fixing pipette, and then the donor cell was injected into the cut line by a flow pressure with an injection pipette to thereby embryo It was made. The obtained embryo was transferred to a TCM199 washing medium, washed 3 times, and then incubated.

Example 3 : Electrofusion and Activation Electrocell Manipulator (ECM 2001, B
The embryos were electrofused and activated using (TX, USA). That is, 0.5 mM HEPES buffer solution (pH 7.2) was added to 0.1 mM MgSO ₄ , 0.05% B.
15 μl of a mannitol solution in which SA and 0.28 M mannitol were dissolved was added to TCM199 washing medium containing embryos with a washing mouse pipette and incubated for 1 minute. Then, the embryo was injected into the mannitol solution added with TCM199 washing medium with a washing mouse pipette, incubated for 1 minute, and the embryo was put into the mannitol solution with a washing mouse pipette. Then, the chamber (3.2 mm chamber No. 453) of the electrocell manipulator was filled with a mannitol solution containing TCM199 washing medium, and then the embryo was placed in the chamber so that the donor cells faced to the cathode. Voltage 0.75 to 2.00 kV / cm, time 15μ
s, the embryos were electrofused by passing a direct current under the condition of twice at 1 second intervals. The fused embryo was immersed in a mannitol solution and washed 3 times with TCM199 washing medium.

1 mg of ionomycin (Sigma Chemical Co., USA) in 1.34 ml of DMSO
Were dissolved and TCM199 wash medium containing 1% BSA was added until the final concentration of ionomycin was 5 μM, and the electrofused embryos in the resulting ionomycin solution were activated by incubating in the dark for 4 minutes. . The ionomycin was then removed by incubating the activated embryos for 5 minutes in a 35 mm dish into which TCM199 wash medium containing 10% fetal calf serum was dispensed.

Example 4 : Post-activation of fused embryos and in vitro culture Cycloheximide (10 mg / ml of ethanol) is added to mTALP (see Table 3) of the in vitro culture medium to a final concentration of 10 μg / ml. The activated embryo was cultured in 25 μl of the obtained cycloheximide solution for 4 hours and then activated. Then, the selected embryo was placed in mTALP and cultured under the conditions of 39 ° C. and 5% CO ₂ for 1 week.

By the above method, the present inventors have used SNU5 (Kore5) to clone tiger embryos produced using tiger ear cells as nuclear donors and Korean cattle as recipient oocytes.
An Tiger NT Embryo) and KCTC (Korean Colle) on March 10, 2000
ction for Type Cultures; KRIBB # 52)) as KCTC0752BP.

Example 5 : Production of embryos using feline-derived oocytes Methods similar to those in Examples 1 to 4 above, except that feline-derived oocytes were used as recipient oocytes The embryo was produced by and cultured.

Example 6 : Cryopreservation, thawing and transplantation of embryos The embryos obtained from Examples 4 and 5 were cryopreserved for long-term storage.
First, the freezing medium (see Tables 9 and 10) is dispensed into a 35 mm dish, and the freezer is moved to maintain it at -5 ° C. The embryos to be frozen were selected, washed with PBS containing 10% fetal bovine serum, placed in freezing medium and incubated for 20 minutes. Then, two air layers were formed on both ends of a 0.25 ml French straw, and a freezing medium containing an embryo therein was sucked to draw the embryo into the straw, and then heated forceps ( Both ends were heat-sealed using forcep). Then, the straw was attached to the freezer, kept at −5 ° C. for 5 minutes, and the lower end of the straw was lightly lifted by the forceps precooled with liquid nitrogen to be seeded. -0.3 after seeding
The temperature was lowered to −30 ° C. at a rate of ° C./min, maintained for 10 minutes, and then frozen and stored in a liquid nitrogen tank.

[0049]

[Table 9] Freezing PBS

[0050]

[Table 10] Freezing medium

In order to thaw the cryopreserved embryo as described above, first, a thaw medium containing PBS containing 20% fetal calf serum was dispensed into a 35 mm dish, and glycerol was added to each to make the glycerol concentration 0. %, 3% and 6% (see Tables 9 and 11). Then, the frozen straw was taken out of the liquid nitrogen tank, exposed to the air for 5 seconds, and then immersed in hot water at 30 ° C. contained in a container having a diameter of 20 cm or more to melt. Next, the air layers at both tips of the straw were cut, the medium containing the embryo was collected, and the embryo was confirmed by a microscope. Thereafter, the embryo freezing medium was removed by sequentially incubating the embryos with 6% glycerol thawing medium, then with 3% glycerol thawing medium, and then with 0% glycerol thawing medium for 5 minutes each.

[0052]

[Table 11] Thawing medium

The above-mentioned embryo was placed in PBS containing 20% fetal bovine serum, sucked into a straw, and then transplanted into the uterus of a surrogate mother animal.

Example 7 : Comparison of Embryos with Different Recipient Cells The embryos obtained from Examples 4 and 5 were transplanted into surrogate mothers in order to examine the difference in embryos depending on the type of recipient oocyte. Then, the number of electrofused oocytes, electrofusion rate (%), division rate (%), number of 8-cell stage embryos (%), number of 16 cell stage embryos (%), 3
A comparison was made in terms of the number of 2-cell stage embryos (%), morula / blastocyst development rate (%), number of embryos transferred, and number of pregnancy after transplantation (see Table 12). At this time, mulberry /
The number of blastocysts is the ratio of embryos that have developed to the stage immediately before transfer to a surrogate mother in the in vitro culture of embryos produced by nuclear transfer.

[0055]

[Table 12] Comparison of cloned tiger embryos

As can be seen from Table 12 above, the development rate and the possibility of pregnancy were higher when the bovine oocyte was used as the recipient than when the cat oocyte was used as the recipient. This is due to the fact that sufficient research has been conducted and optimal conditions have been established for the use of bovine oocytes as a recipient, but that for feline oocytes as a recipient. Is analyzed because it was not possible to establish optimal conditions due to insufficient research.

However, in the conventional research, although a heterogeneous nuclear transfer was carried out using a commercial animal with a large amount of research results, offspring could not be obtained.
Since somatic cloned progeny could be obtained by the technique of heterologous nuclear transfer of wild animals, which has not been sufficiently studied, the present invention makes a significant progress in the production of somatic cloned animals. It is evaluated as achieved.

As described in detail and proved above, the present invention provides a method for producing cloned tigers by the xenogeneic nuclear transfer technique of fusing somatic cells of tigers with oocytes of cattle or cats, clones produced by the above method. Providing tiger embryos and cloned tigers. According to the present invention, one of the methods for preserving wild animals is to permanently transfer the genetic traits of rare or endangered wild animals by carrying out heterologous nuclear transfer to produce cloned fertilized eggs of wild animals. It is expected that it can be widely used in other related fields by using the technique of nuclear transfer between different species.

Various modifications of the invention, in addition to those disclosed herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0060]

[Brief description of drawings]

The above-mentioned objects and configurations of the present invention will be further clarified by the following attached drawings and description.

[Figure 1]   It is a photograph of a donor somatic cell.

FIG. 2 is a photograph showing a process of incising the zona pellucida of a recipient oocyte with a fixing pipette and an incision pipette.

[Figure 3]   It is a photograph showing a process of removing the first polar body and the nucleus from the recipient oocyte.

FIG. 4 is a photograph showing a process of transplanting somatic cells into enucleated oocytes with a fixing pipette and an injection pipette.

─────────────────────────────────────────────────── ─── Continuation of the front page (31) Priority claim number 1999/26165 (32) Priority date June 30, 1999 (June 30, 1999) (33) Country of priority claim Korea (KR) (31) Priority claim number 1999/26166 (32) Priority date June 30, 1999 (June 30, 1999) (33) Country of priority claim Korea (KR) (31) Priority claim number 1999/36373 (32) Priority date August 30, 1999 (August 30, 1999) (33) Priority claiming country Korea (KR) (31) Priority claim number 2000/4381 (32) Priority date January 28, 2000 ( January 28, 2000 (33) Country claiming priority (KR) (31) Number claiming priority 2000/4382 (32) Priority date January 28, 2000 (January 28, 2000) (33) Priority Claiming country Korea (KR) (81) Designated country EP (AT, BE, CH, CY, E, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG) , KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ , DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, O, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Lo, San-ho South Korea 456-360 Kung-ghi-do, Ansan-si, Danwan-dong, Daeuk-Kungnam Apartment 205-601 (72) Inventor Lin, Jeon-mook South Korea 138-040 Seoul, Songpa -Gu, Poonup-Dong 219, Miseon Apartment 1-601 (72) Inventor Park, John-im Republic of Korea 156-032 Seoul, Dongjak-gu, Sandeau 2-Dong 29-1 (72) Inventor Cho, John -Ki 134-032 Seoul, Kandong, Seongnae 2-Dong 230-4 (72) Inventor Kim, Kyoung Korea 151-019 Seoul, Wanaku, Sirim 9-do Gong, Konyang Sirim 5 Chiya Apartment 1205 (72) Inventor Lee, Eun-sung Republic of Korea 200-180 Kangwon-do, Choeng Cheong-si, Soksa-dong 721-2, Soksa Daew Apartment 205-601 (72) Invention Sin-soo-jun South Korea 143-192 Seoul, Wanjin-gu, Jayang 2-Dong 643-13 (72) Inventor Kim, San-ki South Korea 445-920 Kyungi-do, Wasan-gun, Hyangnam-mun, Bunchuk Kuri, Hyang Nam Apartment 1-302 (72) Inventor Sung, Kil-Yang South Korea 131-206 Seoul, June Lang, Myeong Mok 6-Dong, 70-41 (72) Inventor Yong, Hwang- Yul Republic of Korea 440-210 Kung-ghi Daw, Suwon-si, Chang-gang, Song-jok-dong, 85-1 (72) Inventor Anne, Gok-gee Republic of Korea 506-301 Kwangju, Kwangsang, Docheon-Dong 290, Line Apartment 107-102 (72) Inventor Hyun, Sun-Hwan South Korea 690-032 Jeju-do, Jeju-si, Songdo 2-Dong , 1051-1 (72) Inventor Lee, Byung-dong South Korea 449-840 Kyungi-do, Yoon-Insi, Soji-Eup, Poon Dak-Chon-Li, 6-Ga, Woosun Apartment 605-606 (72) Invention Person One, Woo-suk Republic of Korea 135-010 Seoul, Gangnam-gu, Nonhyung-dong 11-2 F term (reference) 4B024 AA10 CA20 DA02 GA03 GA10 GA18 HA20 4B065 AA90X AA90Y AB04 AC20 BA08 BB02 BB08 BB15 BB37 CA60 [Continued summary] ]

Claims (14)

[Claims] 1. A method for producing a cloned tiger, comprising: (I) culturing somatic cells obtained from the tiger to prepare a donor somatic cell line; (II) immature oocytes from bovine or feline ovaries. (III) After removing the cumulus cells surrounding the mature oocyte, a part of the zona pellucida of the mature oocyte is incised to make a cut, and the cut is made. Producing a enucleated recipient oocyte by removing a part of the cytoplasm containing the first polar body from (IV) injecting the donor cell into the enucleated recipient oocyte Transferring the nucleus to the recipient oocyte, followed by electrofusion to activate the electrofused cell to obtain an embryo; (V) post-activation of the activated embryo in vitro And then in vitro culturing; (VI) in vitro culturing By transplanting the embryo into a surrogate mother animal, step of birth clones tiger; method of producing clones tiger comprising. 2. The somatic cell line prepared in step (I) is uterine perfusate of tiger, endometrium, fallopian tube, cells isolated from ear or muscle, cumulus cell or fetal fibroblast cell. The method for producing a cloned tiger according to claim 1, wherein 3. The method for producing a cloned tiger according to claim 1, wherein the somatic cell line of step (I) is preserved by subculture, serum starvation culture or freezing. 4. The cumulus cell surrounding the oocyte of step (III) is characterized by being physically removed with an exposing pipette after treating the oocyte with hyaluronidase. Method of producing cloned tiger. 5. The enucleation of the oocyte in step (III), the incision is made by making an incision in the zona pellucida of the oocyte with a micromanipulator, and the oocyte is arranged by orienting the incision vertically. The whole cytoplasm of the oocyte is fixed by fixing the lower part of the oocyte using a fixing pipette and holding it so that it does not move, and then gently pressing it from above the oocyte using an incision pipette. The method for producing a cloned tiger according to claim 1, which is carried out by removing the cytoplasm containing the first polar body in an amount corresponding to 10% to 15% from the cut. 6. The nuclear transfer of step (IV) is carried out by injecting donor cells into recipient oocytes enucleated from cuts on the zona pellucida of the oocytes, The method for producing a cloned tiger according to claim 1. 7. The step (IV) of electrofusion is performed at a voltage of 0.75 kV / each time.
The method for producing cloned tiger according to claim 1, wherein the method is carried out by applying a direct current under the conditions of cm-2.00 kV / cm, time of 15 μs, and one second interval twice. 8. The production of the cloned tiger according to claim 1, wherein the activation in step (IV) is carried out simultaneously with electrofusion under the condition that the electrofusion is carried out in a medium containing Ca ^2+. Method. 9. The method according to claim 1, characterized in that the activation in step (IV) is carried out in an ionomycin solution in the dark, provided that the electrofusion is carried out in a Ca ²⁺ -free medium. Production method of clone tiger. 10. The cloned tiger according to claim 1, wherein the post-activation in step (V) is performed by culturing the embryo in a cycloheximide solution or a DMAP (4-dimethylaminopurine) solution. Production method. 11. The step (V) in vitro culture of the post-activated embryos to mTALP
The method for producing cloned tiger according to claim 1, which is performed by culturing in a medium, mSOF medium or mCR2aa medium. 12. The in vitro cultured embryo of step (V) is treated with penicillin-streptomycin, CaCl ₂ , glucose, MgCl ₂ , Na pyruvate and PB.
The method for producing a cloned tiger according to claim 1, further comprising a step of preserving by freezing in a freezing solution containing S, and then thawing the freezing solution before use. 13. The steps (I) to (V) according to claim 1, wherein a tiger ear cell and a bovine (Bos taurus coreanae) oocyte are used as a nuclear donor cell and a recipient oocyte, respectively. SNU5 (Korean T
iger NT Embryo, KCTC 0752BP). 14. The SNU5 (Korean Tiger NT Embryo, KC according to claim 13
A cloned tiger produced by the method of step (VI) according to claim 1, using TC0752BP) as an embryo.