KR20010069167A - Embryo from wild animal with inter-species nuclear transplantation and method for production thereof - Google Patents

Abstract

PURPOSE: A nuclear transplantation method between different species and a method for producing somatic cell cloned oocyte of wild animal are provided. The method can be applied to various other mammal animals including human. CONSTITUTION: The method for producing somatic cell cloned oocyte of wild animal comprises the steps of: collecting an immatured oocyte from a cow and incubating it; separating cell lines from various tissue of a tiger; cutting the transparent membrane of the matured oocyte and removing fist polar body and 10 to 30% of cytoplasm; transplanting the cell lines as a donor into the nucleus removed oocyte; adding the DC electric shock into the transplanted oocyte to produce the fusion cell; activating the fusion cell in a medium containing chemical materials immediately after electric shock; and second activating and incubating the fusion cell in a drop medium.

Description

Embryo from wild animal with inter-species nuclear transplantation and method for production

The present invention relates to a method for producing cloned fertilized eggs in wild animals, and more particularly, by somatic cloning by collecting and denuclearizing immature eggs derived from bovine through nuclear transplantation technology and transplanting nuclei of somatic cells isolated from wild animal tissue. To provide a method for producing cloned fertilized eggs of wild animals. According to one preferred embodiment of the present invention there is provided a gestational cloned embryo of a tiger produced by transplanting the nucleus of somatic cells isolated from tiger ear tissue into a bovine-derived nucleus egg.

An object of the present invention is to produce a cloned fertilized egg of a wild animal that can be conceived by somatic cloning by performing a heterologous nuclear transfer between a nucleus of a wild animal and a nucleus of a bovine egg using a nucleus of a bovine egg.

According to the present invention, there is provided a method for producing a cloned fertilized egg of a tiger, which consists of the following process steps:

(a) harvesting immature eggs from slaughtered ovaries;

(b) harvesting the body tissue of the tiger, separating the cell line;

(c) dissecting a portion of the zona pellucida of the mature egg prepared in step (a) and denuclearizing by removing 10-30% of the cytoplasm;

(d) transplanting the donor cells prepared in step (b) into denucleated eggs of step (c);

(e) electrically fusion of the embryonated eggs implanted and adhered in step (d) to a voltage under DC conditions;

(f) activating the fused cells concurrently with fusion or in a medium to which chemicals have been added; And

(g) culturing the activated cells in post-activation and micromedia.

According to one embodiment of the invention, the cell of step (b) is a cell isolated from the tiger's ear.

In addition, the preservation of the step (b) can be made by subculture, serum starvation or cryopreservation.

Hereinafter, the preferred examples and experimental examples performed according to the method of the present invention will be described in detail.

Example 1 Nuclear Transplantation

The nuclear transfer preparation process was performed as follows.

Step 1: Preparation of the Micro Pipette

In preparing the micro pipette,

Puller (Narishige PC-10), Microforge (Narishige MF-9), Glinder (Narishige EG-4).

First, in order to manufacture incision and denuclearization pipettes, a weight (1 light, 1 heavy or 2 light, 1 heavy) was used using a glass tube (Drummond; Sigma, P-2174 or narishige). After attaching and fixing to Step 2 with a pulling step, adjust the temperature (heat 1: 101 ℃, heat 2: 101.5 ℃).

Second, remove the weight (1 light) or weight using a glass tube (Drummond; Sigma, P-2174 or narishige) for the manufacture of injection pipettes (Injection pipette, internal diameter 30-40 ㎛) Fix in Step 2 and adjust the temperature (heat 1: 101 ℃, heat 2: 101.5 ℃).

Next, to prepare a holding pipette (Holding pipette, inner diameter 70 ~ 130㎛) (two) was carried out as follows.

i) When using glass tubes (Drummond; Sigma, P-2174):

Attach the weight (1 light ~ 2 light, 1 heavy), fix it to the pulling step (Step 1), and adjust the temperature (heat 2: 101.5 ℃).

ii) When using glass tubes (Narishige G-1 or GD-1)

Attach the weight (1 light, 1 heavy), fix it to the pulling step (Step 1), and adjust the temperature (heat 2: 101.5 ℃).

Fourthly, for the manufacture of pipettes for cleaning and transporting eggs

The alcohol lamp was used to have an inner diameter of 300 µm or more, and once used, it was discarded.

Fifth, for the manufacture of pipettes for removal of cumulus cells (for denuding)

An alcohol lamp was used to prepare a plurality of inner diameters of about 130 μm.

Second Step: Preparation of Culture Solution

Culture medium was prepared as follows.

TCM-199 for IVM is manufactured in the same way as in vitro fertilization, and on the day of use with TCM-W, a small amount for work is prepared, but 1ml of FBS (FCS) is added to 9ml of TCM-W to make 10% FBS TCM-W. Using a 100µl pipette, use 20µl droplets in a suitable size (35mm, 60mm, 100mm, etc.) dish, apply with mineral oil, and use.

In addition, in order to prepare a hyaluronidase (Sigma, H-3506) solution, 5ml of TCM-W was added to 0.0500g of hyaluronidase (Hyaluronidase) to make a stock (stock). The prepared stock solution was dispensed into eppendorf test tubes by 111 μl and stored in the freezer. In use, 1 ml of TCM-W is dispensed and the final concentration is adjusted to 0.1% and dispensed into 35 mm dishes for dendritic cell removal.

And, for the preparation of Cytochalasin B (sigma, C-6762) solution,

After dissolving 1 mg of cytocalasin B in 133 μl of DMSO (Sigma, D-5879), the concentration is 7.5 mg / ml. Dispense 1 μl into eppendorf test tubes and store in the freezer. In use, 1 ml of 10% FBS (or FCS) -added TCM-W is added and used for microscopic cutting and enucleation.

In addition, PHA-P (Phytohematoglutinin: L-9132) solution is prepared by mixing 5 mg of PHA with 10 ml of TCM-W and dispensing 100 μl into eppendorf test tubes, and then 400 μl of TCM-W into eppendorf test tubes when used in the experiment. Use it to make working microdroplets by adding. At this time, the final concentration is to be 100 ㎍ / ml.

And, in order to prepare the electrofusion and activation media (Electrofusion and Activation media) the amount of mannitol (Sigma, M-1902) added according to the Mole concentration (100ml standard) 0.28M mannitol: 5.1016g; 0.25 M mannitol: 4.555 g; 0.26 M mannitol: 4.7372 g; 0.1 mM MgSO ₄ , 0.1 mM CaCl ₂ , other additives (regardless of mannitol mole concentration): 0.1 mM MgSO ₄ or MgCl ₂ , 0.1 mM CaCl ₂ (added in the fusion and active co-group); Hepes 0.5 mM; Add 0.05% BSA but add up to 100 ml DW.

Adjust the pH to 7.2-7.4, dispense about 4ml into 5ml test tube and store in freezer (long-term) or freezer (short-term).

On the other hand, in the manufacturing method of DMAP (Sigma, D-2629), 0.0031 g DMAP is added to a 10 ml IVC solution, dissolved and filtered (using a 0.22 μm filter), and then dispensed into a eppendort test tube and frozen (1.9 mM).

Cycloheximide (Sigma, C-7698) is prepared by dissolving 1 g of Cycloheximide in 100 ml of ethanol and adding a 1000 × stock to the IVC medium to a final concentration of 10 µg / ml.

In addition, Ionomycin (Sigma, I-0634) is dissolved in 1mg of Ionomycin in 1.34ml of DMSO (Sigma, D-5879) to prepare a 1mM stock solution, and then 1% BSA TCM-W or IVC medium to a final concentration of 5㎛ It is added and used in the container, and the stock solution is kept frozen.

Ca ²⁺ Ionophore (A23187) is dissolved in 1 mg of Ca ²⁺ Ionophore (A23187) in 1.66 ml of DMSO (Sigma, D-5879), so that 1% BSA, TCM-W or IVC is used to prepare a final concentration of 5 μm. Add to medium and store the stock solution for freezing.

For culture medium, CR2aa was used (No. 2), and Hoechst 33342 (Sigma, B-2261) was diluted with 5 ml of distilled water (25 mg / ml stock) in 25 mg of Hoechst, and then 1 to 2 µl of stock. Dispense and freeze into eppendorf test tubes. In addition, 10% FBS is added to a test tube, 1 ml of TCM-W is diluted, and it is used after use, and it uses (final concentration 5-10microgram / ml).

After standing for 10-15 minutes, denuclearization can be determined using a dark room or a fluorescence microscope.

Nocodazole stock (Sigma, M-1404) was diluted in 2.5 ml of DMSO (Sigma, D-5879) in a 10 mg nocodazole bottle, and then divided into 50 to 100 μl in eppendorf test tubes (4 mg / ml stock). [Mu] l is added (0.4 [mu] g / m) to 10 ml of the desired culture.

Acid Tyrode's (Sigma, T-1788) manufacturing method is Embryo-tested finished product (25µl) is used after oil coating.

Step 3: preparing a dish for undifferentiated microscope work

Differential Interference Contrast (DIC) microscope work dishes were prepared as follows.

Perforate the 50mm dish (Falcon 1006) with a diameter of 1 ~ 3cm, apply Silicon (ShinEtsu KE45) around the hole inside the dish, and attach the cover glass of 2 × 4cm in width x length on the silicone .

Finally, rinse with alcohol.

Step 4: Microfabrication for DIC Microscope

The microfabrication method for a DIC microscope was produced as follows.

Dispense 5-10 μl micro dropwise vertically using a 20 μl pipette on the cover glass of the DIC working dish (the number of micro drop counts 1 per 20-30 eggs) and then use a 1 ml pipette Oil (sigma, M-3516) is applied around the microspheres. At this time, microdrops are formed with a medium suitable for each process.

Step 5: Prepare Somatic Cells for Donor Nuclei

Somatic cell line for donor preparation was performed as follows.

Cell lines were isolated from tiger tissue (see Tissue).

Experimental Example 1 Cell Separation from Ear

Shave the hair around the area to be removed from the ear tip of an adult or young animal and disinfect with rubbing alcohol and betadine. After dissecting 1 ~ 2cm ² of skin tissue with a sterile instrument, prepare PBS solution in 50 ml test tube, put the excised tissue and store it at 4 ° C and transport it to the laboratory.

After washing with PBS solution again, and fixed with sterile forceps, skin and cartilage tissue, including body hair, are separated by sterile scissors and blades, and tissues inside the skin facing the cartilage tissue are collected.

The tissue piece was washed again with PBS solution, and then cut into blades, and a mixture of 0.25% trypsin, 1 mM EDTA solution and 0.8% Collagenase type II (GIBCO BRL, Life Technologies, USA) solution was added. Incubated in a saturated humidity incubator at 39 ° C. and 5% CO ₂ .

After standing, it is transferred to a centrifuge test tube, and after vortexing and pipetting, PBS solution is added and centrifuged under the same conditions.

After final centrifugation, the precipitated cells were suspended in 10% FCS, 1% non-essential amino acid solution, P / S added DMEM (Dulbecco's Modified Eagles medium, GIBCO BRL, Life Technologies, USA) and transferred to Petri dishes for cell culture. Incubate in a saturated humidity incubator at 39 ° C., 5% CO ₂ .

When cells adhere to the bottom of the culture plate and divide and proliferate to form a monolayer, this is regarded as the establishment of a primary cell line.

Experimental Example 2 Passage Culture

Subculture of the primary cell line in which the monolayer was formed was performed as follows.

The culture medium of Petri dish was discarded and 0.25% trypsin and 1 mM EDTA solution of Experimental Example 1 were added thereto, and after 1 to 2 minutes, the culture medium was replaced.

The cells are resuspended in culture medium by pipetting, then aliquoted into fresh Petri dishes, and then cultured in a 39 ° C., 5% CO ₂ saturated humidity incubator.

Experimental Example 3 Serum Starvation Culture

10% FCS 1% non-essential amino acid solution of Experimental Example 1, P / S addition cultured in DMEM (Dulbecco's Modified Eagles Medium, GIBCO BRL, Life Technologies, USA), replacing the proliferating cell line with 0.5% FCS addition culture, After 3 to 10 days of incubation it was provided as a donor nucleus.

Experimental Example 4: Cryopreservation of Cell Lines

Resuspend the adherent cells in the same manner as in Experimental Example to adjust the number of cells to 1 X 10 ⁵ / ml, and then to make 10% concentration of DMSO (Dimethyl sulfoxide, Sigma, USA) in the total culture solution containing the cells. After dilution, the solution is enclosed in Cryovial (Corning, USA).

Store Cryovial in -70 ℃ freezer for 18-24 hours and store in liquid nitrogen tank.

Experimental Example 5: Fusion of cryopreserved cell lines

Cryovial was removed from the liquid nitrogen tank and fused in hot water at 37 ° C., and the fused cells were placed in a test tube for centrifugation. PBS was added and centrifuged at 1500 rpm for 2 minutes. Discard the supernatant and carefully centrifuge twice with careful cell precipitate.

After final centrifugation, the precipitated cells were suspended in 10% FCS, 1% non-essential amino acid solution, P / S added DMEM (Dulbecco's Modified Eagles medium, GIBCO BRL, Life Technologies, USA) and transferred to Petri dishes for cell culture. Incubate in a saturated humidity incubator at 39 ° C., 5% CO ₂ .

Experimental Example 6: nuclear observation of somatic cells (using Giemsa Stain)

The following preparations were used for nuclear observation of somatic cells using Giemsa stain. That is, a stock solution (0.9% sodium citrate solution or 0.56% KCL solution); Fixative (methanol: acetic acid = 3: 1); 5% Giemsa; Slide glass; 15ml test tube

In the process of observing somatic cells,

After floating the cells in the dish (Single cell), centrifuged for 2 minutes at 1500rpm in a 15ml test tube to obtain a cell pellet (pellet).

Next, the stock solution is added to the pellet, suspended, hydrated for 15 minutes, and then a fixed solution corresponding to 1/3 of the total volume is added.

Then, centrifuged (1500 rpm, 2 minutes), and then washed with a fixed solution (centrifugation → supernatant removal → resuspension, 3 times), leaving a fixed solution to a titration concentration after the last centrifugation and dropping onto a cell suspension slide ( Spread as much as possible, then dry in air.

After dyeing for 15 minutes in Giemsa solution, the excess dye solution is removed from the slide using distilled water.

Then, it observes with high magnification (10x40) at low magnification (4x10).

Example 2 Internuclear (Wildlife) Nuclear Transplantation

Stage 1: In vitro maturation

Medium for in vitro maturation was prepared as follows.

Culture TCM199 (No. 3); For washing-TCM199 (No. 2);

Serum (FBS, dispensed in 1 ml portions); E ₂ (No. 7); FSH (No. 7)

Prepare the culture solution and dispense 0.5 μl E ₂ into a 4-well dish and volatilize the alcohol. Dispense 12.5 μl FSH (NO.7), add 1 mL of FBS to 9 mL of TCM199 for culture, and dispense 0.45 mL into each well. Distilled water is dispensed between the wells using a squeeze bottle. Dispense 2.5 ml of TCM 199 remaining in a 35 mm dish to use as a final wash.

Ovarian collection is prepared by physiological saline (35 ℃), scissors, RP gloves, boots, etc. to collect the ovaries from the slaughterhouse.

To prepare the eggs, prepare a 100mm dish with grid scales of 1cm intervals and a 35mm dish dispensed with 2ml of TCM199 for washing. A 2-6 mm follicle is aspirated from the collected ovary using a 10 ml syringe equipped with an 18G needle.

Next, the egg and washing are performed. First, eggs that are sufficiently adherent and have a homogeneous cytoplasm are selected, and the selected eggs are washed three times, selected in the final culture TCM199, and 35 μl of 20-50 eggs per each well are dispensed.

In vitro maturation is carried out in a 5% CO ₂ incubator for 24 hours.

Second Step: Denuding

Preparation was as follows; Cleaning pipettes, removal pipettes, hyaluronates, micro-manufactured for TCM-W

The procedure was as follows.

IVM 16-22 hours eggs are washed once with TCM-W using a cleaning mouse pipette.

Dispense the egg into the Hyaluronidase solution (No. B-③).

Peel the oocytes with a removal pipette. Wash and let stand 3 times in TCM-W. It is desirable to perform the work quickly.

Step 3: Incision and Denuclearization

Preparation was as follows.

Working dishes (No. C), working micro-cytochalasin B, pipettes: fixation pipettes, cutting & enucleation pipettes

Incision and denucleation process were as follows.

Place the working dish on the manipulator plate and fit the pipette.

After the fixing pipette is mounted on the left arm of the manipulator, the incision pipette is mounted on the right arm of the manipulator.

The direction and position of the pipette is adjusted, for example, as follows.

That is, the fixing pipette is positioned at 9 o'clock and the cutting pipette is positioned at 3 o'clock. Place the pipette adjuster in neutral to adjust the pipette to move freely up, down, left and right. With both pipettes flowing up and down into the working drop, angle the pipette so that it does not touch the edge of the dish and the pipette tip is centered in the micro drop.

About 10-30 oocytes in TCM-W were grouped into one group using a cleaning mouse pipette (more than 200 µm in diameter) and then moved dropwise after incision and denucleation.

The incision was made in the following manner:

That is, focusing on oocytes first using coarse and fine screws, and then moving the two pipettes up and down to adjust the focus. The first polar body is positioned at 12 o'clock of the gripping pipette by moving the two pipettes. The phage pipette is in close contact with the oocyte at 9 o'clock and hydraulically fixed to the oocyte. Puncture the incision pipette at 1 o'clock, and pass through the zona pellucida. Positive pressure is applied to the gripping pipette to separate the oocytes, and the gripping pipette is contacted with the transparent zone of the upper end of the first polar body passing through the incision pipette, and the two pipettes are rubbed to cut the transparent zone.

Denucleation was performed as follows.

Rotate the injured egg to position the incision vertically and place the fixation pipette at the bottom of the egg to prevent the egg from moving downward. Then, press the incision pipette on the egg and press down the cytoplasm, including the first polar body. 30% denuclearization.

Alternatively, the incision of the egg is rotated vertically in the 3 o'clock direction, and then the fixation pipette and the incision pipette are pressed down from above to perform 10-30% denucleation of the cytoplasm including the first polar body. The denuclearized group 1 eggs were washed three times with TCM-W and allowed to stand in TCM 199 (B-①) for IVM until nuclear transfer. Since the egg after denuclearization is very fragile, use a mouse pipette with an inner diameter of at least 300 µm to ensure that the cytoplasm of the egg does not escape through the incision after the operation.

Step 4: Isolation of Single Cells for Donor Nuclei

Discard the culture solution in Petri dishes and let stand for 1 to 2 minutes by adding 0.25% trypsin, 1 mM EDTA solution of Experimental Example 1, pipetting in PBS (-) (GIBCO BRL, Life Technologies, USA) with 1% FCS The cells were resuspended and adjusted to an appropriate number of cells (2,000 / 0.1 ml) and dispensed into Eppendorf tubes.

Step 5: Injection of Donor Cells

Preparation was as follows; Working dish, working drop making-PAH, pipette: fixation pipette, injection pipette, donor cell micro (see Donor cell preparation)

The procedure was as follows.

Place the working dish on the manipulator plate.

The incision pipette is replaced with an injection pipette.

Oocytes in IVM medium are transferred microscopically for injection after one to three washes in a TCM-W drop using a cleaning mouse pipette.

Insert the injection pipette into the donor cell microparticles, inhale one or more donor cells of 20-40 μm in size, and then move the pipette microscopically for injection.

Place the incision perpendicular to both pipettes and the 1 o'clock position and secure with a fixed pipette.

An injection pipette is injected into the incision and hydraulically infused one donor cell.

The resulting nuclear transfer egg group is transferred to a washing microscopically with a mouse pipette, and then washed and left to set once or three times.

Example 4 Cell Fusion

Preparations for cell fusion were as follows; Instrument-BTX ECM 2001

Manitol is dispensed from 0.4 ml to 1 ml into three wells of 4-well dishes (wells 1, 2 and 3, in any order) and 10% TCM- for the other well (4). Prepare mannitol to which W is added.

If fusion and activation are performed at the same time, Ca ²⁺ -added fusion medium is used, and in the activation group after fusion, Ca ²⁺ fusion medium is used.

Next, 0.2-1 ml of mannitol is placed in two electrodes (3.2 mm chamber No. 453) and connected to a BTX-cell manipulator.

At this time, the cell fusion conditions were as follows.

DC condition: Voltage is 0,75 ~ 2.25kv / cm, Times is 1 ~ 2 times, and Seconds is adjusted within 60ms of recovery time × time.

The procedure was as follows.

One hour prior to operation, the BTX was run and preheated, and 10-20 μl of mannitol was sucked into the cleaning mouse pipette and added to the microtransplanted TCM-W microspheres.

After standing for at least 1 minute, a nuclear transfer egg is injected into well 4 (10% TCM-W mannitol) using a cleaning mouse pipette.

After standing for 1 minute or more, the wells are placed in the well 1 using a cleaning mouse pipette, and the nuclear transfer egg is transferred to the second well in the same manner.

Using a mouse pipette, the nuclear transfer eggs are placed between two electrodes one by one and aligned so that donor cells face the positive electrode.

The energized and fused nuclear transfer embryos are collected in three wells and transferred to TCM-W for cleaning three times after the fusion of one group is completed.

After treatment for 30 to 60 minutes in the cytochalasin B microsphere, the group that will perform the fusion and activity at the same time is a postactivation process, and the group to be activated after the fusion is activated using the above chemicals.

Activation was performed as follows.

In the case of induction of activation by the electrofusion method at the same time as the fusion, electrofusion is performed in a Ca ²⁺ -added fusion medium.

In the case of induction of activation by the ionomycin (B-⑨) treatment, B-⑨, incubated for 4 minutes in a 50 µl drop (it is sensitive to the light, blocking the inflow of light as much as possible), and 10% FBS or 30 mg / in 35 mm dish. ml BSA is added and the inomycin is removed under washing medium and left to stand for 5 minutes.

Example 5 Postactivation

About 5-10 eggs were immersed in 25 µl of cyclocloximide for 6 hours, and 5-10 cells were immersed in 25 µl of DMAP for 4 hours.

Cultivation was performed as follows.

Example 1 The culture medium (Culture medium) of the second step is alternatively incubated for 5 to 8 days in a 5% CO ₂ incubator or 5% CO ₂ , 7% O ₂ , 88% N ₂ . After about 24 hours after activation (Activation), it is arranged within the microscopic division by egg phase and development stage.

Every morning and evening, the gumran to observe the subsequent development.

As described above, using a bovine egg as a nucleated egg, isolating and preserving a cell line from various tissues of a tiger which is a wild animal, dissecting a part of the cell membrane of the first cell and denuclearizing 10-30% of the cytoplasm, and then the donor cells By implanting the nucleus into the denuclearized egg and electrofusion, and after the activation and post-activation step, by culturing and maturation, it is possible to produce fertilized tiger embryos capable of replicating somatic cells as part of wildlife conservation.

Hereinafter, the types and compositions of the various media used in each of the above-described processes of the present invention are shown in Tables 1 and 2, and Table 3 shows the development after creation of tiger cloned fertilized eggs.

The 8- cell fertilized egg produced in the present invention was named Bos taurus coreanae embryos / TcEar and was deposited as KCTC 0719BP at the Biotechnology Research Institute of Biotechnology on January 7, 2000.

TCM-Culture medium ingredient Concentration TCM liquidNa-pyruvateP / S Gibco 11150-0591mM1% (Penicilin 10000IU, Streptomycin 10mg)

CR2aa medium ingredient Develope I Develope II CR-Wash NaCl ^o 114mM 114mM 114mM KCl ^o 3.1mM 3.1mM 3.1mM NaHCO ₃ ^O 25mM 25mM 2 mM NaH ₂ PO ₄ 0.35mM 0.35mM 0.34mM Sod.Lactate ^r 15 mM 15 mM 15 mM CaCl ₂ · 2H ₂ O ^d 2 mM - 2 mM MgCl ₂ · 6H ₂ O ^d 0.5mM 0.5mM 0.5mM EAA - One% - NEAA One% One% One% Insulin One% One% One% Glutamine - 1 mM One Glycine 0.37mM 0.37mM 0.37mM Citrate 0.33mM 0.33mM 0.33mM HEPES - - 10.5mM Na-pyruvate 0.3mM 0.3mM - Glucose - 1.5mM - Phenol-red 10 μg / ℓ 10 μg / ℓ 10 μg / ℓ BSA 3mg / ml - 3 / ml FCS - 10% - Kanamycin 0.75 µg / ml 0.75 µg / ml 0.75 µg / ml

Development after tiger cloning fertilization Number of fused eggs % Convergence % Split 2cell 4 to 7cell 8cell Number of blastocysts 272 139 (51) 96 (73) 20 19 23 0

As can be seen from the above examples and experimental examples, according to the present invention, as a method of preservation of wild animals, it is possible to produce cloned fertilized eggs of wild animals using nuclear transplantation, which will be reproduced in the field of animal husbandry and medicine in the future. There is an effect of mass production of animals and drugs or organs using the same. In addition, there is the possibility of producing cloned animals according to the present invention and pioneering applications related thereto, and thus the present invention is a very useful invention in animal husbandry, medicine and animal cloning industry.

Claims (3)

In the production method of cloned fertilized eggs of tiger, (a) harvesting and culturing immature eggs from slaughterhouses; (b) separating and conserving cell lines from various tissues of the tiger's body; (c) dissecting a portion of the zona pellucida of the mature egg prepared in step (a) and denuclearizing by removing 10-30% of the cytoplasm with the first polar body; (d) transplanting the donor cells prepared in step (b) into denucleated eggs of step (c); (e) electrically fusion of the embryonated eggs implanted and adhered in step (d) to a voltage under DC conditions; (f) activating the fused cells concurrently with fusion or in a medium to which chemicals have been added; And (g) a method for producing cloned fertilized eggs, comprising activating the activated cells and culturing them in post-activation and micro medium. The method of claim 1, wherein the cell line of step (b) is a cell line isolated from tiger ears. Cloning fertilization of tiger produced according to claim 1 or 2 Bos taurus coreanae embryos / TcEar (KCTC 0719BP).