KR100862298B1 - Dual pipet and method for enucleation, nuclear transplantation and somatic animal clon production by using the same - Google Patents

Abstract

The present invention relates to a double pipette used for the production of somatic cell replication embryos consisting of an incision pipette and a denuclearization / nuclear transplant pipette, a method for denuclearization / nuclear transplantation of nucleated oocytes using the same, and a method for producing somatic cloned animals.

The double pipette of the present invention is a micromanipulation pipette in which an incision pipette and a denuclearization / nuclear transfer pipette are coupled to each other, and by using the double pipette, the rate of denuclearization in the nucleus-nucleated oocytes during the production of the somatic cell embryo and the blastocyst development rate of the fertilized egg afterwards are used. It shows an increasing effect.

Therefore, the double pipette of the present invention and the method for denuclearization and nuclear transfer of nucleated oocytes using the same can be usefully used for the production of somatic clones.

Description

Dual pipet and method for enucleation, nuclear transplantation and somatic animal clon production by using the same}

1 is a cross-sectional view of the cutting pipette 10 of the present invention.

2 is a cross-sectional view of the denuclearization / nuclear transfer pipette 20 of the present invention.

3 is a front and partial enlarged view of a double pipette 100 of the present invention.

4 is a photograph of the double pipette 100 of the present invention in which the incision pipette 10 and the denuclearization / nuclear transfer pipette 20 are combined and fixed.

5 is a photograph of the case of using the cutting pipette 10 and the denuclearization / nuclear transfer pipette 20 of the double pipette 100 of the present invention in one working drop.

Figure 6 is a diagram showing the denuclearization of the first polar body and the chromosome- spindle complex with a denuclearization / nuclear transfer pipette on the nucleus ova after making an incision with the incision pipette of the double pipette of the present invention.

Figure 7 is a diagram showing a state of denuclearization by the conventional squeegee method.

Figure 8 is a diagram showing a state of denuclearization by giving a negative pressure to the nucleus nucleus with a pipette for fixing the nucleus nucleus.

9 is a diagram showing a state in which the somatic cell donor nucleus 19 is injected into the nucleated nucleus egg 3 denuclearized with the denuclearization / nuclear transfer pipette 20.

10 is a view showing the injection of the somatic cell donor nucleus 9 into the nucleated nucleated oocytes 3 denuclearized by the nucleus oocyte fixation pipette 50 or the squeezing method.

Fig. 11 is a diagram showing the state of electrofusion of nucleated oocytes 3 into which somatic donor nuclei 9 are injected.

12 is a view showing the appearance of differentiated somatic cell cloned egg after the working step.

Figure 13 is a diagram showing the appearance of somatic cell replication of the blastocyst stage.

<Explanation of symbols for the main parts of the drawings>

3: egg core 6: first polar body

8: Chromosome- spindle spindle 9: Donor nucleus

10: incision pipette 11: capillary

12: Closed tip end 13: Closed tip feature

20: Pipette for denuclearization and nuclear transfer 21: Capillary tube

22: open tip end 23: open tip shape

50: fixed pipette 100: double pipette

The present invention relates to a double pipette used for the production of somatic cell replication embryos consisting of an incision pipette and a denuclearization / nuclear transplant pipette, a method for denuclearization / nuclear transplantation of nucleated oocytes using the same, and a method for producing somatic cloned animals.

Somatic cloning technology has a wide range of applications such as securing high quality individuals, producing high value-added bioreactor animals, producing disease model animals, producing human donor animals, and preserving endangered animals. In addition, the application of somatic cloning technology enables the establishment of embryonic stem cell lines in patient somatic cell-derived blastocysts, and in the case of differentiating cells essential for treatment from the embryonic stem cells thus established, the patient's immune rejection response can be avoided. Clinical availability is high (Trounson A., Reprod Fertil Dev 10 : 121-125 (1998)).

The basis of this somatic cloning technique is the fusion of donor nucleus somatic cells with divalent chromosomes and nucleated nuclei with genetic material removed. Nucleated oocytes to which the donor nucleus is to be transplanted must have the ability to develop after fusion with somatic cells. To develop this capacity, the embryos develop until the second stage of meiosis (when the egg is naturally ovulated in the body). Advanced nucleated egg should be used. For this purpose, artificially cultivate the egg in vitro, or hormonal treatment to develop the egg in the body is used.

The recovered eggs undergo denuclearization prior to fusion with the somatic cells, and are usually removed using a physical method to remove the nucleus of the egg (the cytoplasm of the second meiosis midboard).

The rate of denuclearization at the time of nuclear transfer and denuclearization is an important variable in somatic cloning technology. Especially, in the case of a very limited number of animals or an animal that is difficult to obtain eggs, the effective use of eggs is directly related to individual production. Effective denuclearization is very important.

The denuclearization method of nucleated nucleus of the nucleus has been known so far by special treatment such as squeezing and aspiration using micromanipulators in the state of treating the egg with a drug such as cyto-calacin to prevent cytoskeletal damage existing in the cell. Is made by.

Nevertheless, in the denuclearization process, cytoplasmic shock and loss of some organelles by mechanical manipulation, cytoplasmic loss associated with the removal of nuclear genetic material, and damage to the cytoskeletal structure distributed throughout the zona pellucida, the cell membrane and the ovum may be caused. In addition, in terms of function, the nuclear-cytoplasmic signal transduction system may be disrupted by abrupt nuclear removal, which may result in fatal damage to embryo development.

According to one report, the chromosome-spindle complexes of bovine eggs matured in vitro for 20 hours are often not close to the first polar body, and the efficiency is only 59% when denuclearization by inhalation method (Bordignon V., Smith LC. , Mol Reprod Dev 49 : 29-36 (1998); Mohamed Nour MS., Takahashi Y., Theriogenology 51 : 661-666 (1999)).

In order to solve such a problem, the following denuclearization method has been developed and used.

First is the increase in denuclearization efficiency through activation. As a result of comparing the denuclearization rate before and after activation of the first meiosis mid-aged mature egg, the denuclearization rate of the activated egg was significantly higher (91.5% vs 59.9%; Mohamed Nour MS., Takahashi Y., Theriogenology 51 661-666 (1999).

Second, centrifugation induces stratification of egg organelles, followed by centrifugation with a percoll density gradient, and separation of the chromosomal- spindle spindle complex from the egg cytoplasm (Tatham BG., Dowsing AT., Trounson AO., Biol Reprod 51 : 661-66 (1995).

Third, before denuclearization, demicolcine is treated to induce genetic material to the egg surface and denuclearize by inhalation method (Baguisi A., Biol). Reprod 67: 442-446 (2002).

Fourth, the squeezing method of squeezing the nucleus after puncturing a small hole in the egg by efficiently constructing the fixation and incision angles of mature eggs using a fixing pipette and an incision pipette (see Republic of Korea Patent No. 342437) And fifth, there is a method of instantaneously irradiating a laser precisely aimed at the chromosome- spindle spindle complex of the egg together with the first polar body.

However, even when the efficiency of mechanical denuclearization technology is improved by using the above method, the following problems occur, and the improvement of the fundamental efficiency is limited by these problems.

First, the activation method may cause cell cycle inconsistency between nucleophiles and donor cells, resulting in chromosomal drainage abnormalities during mitosis after the first DNA replication after fusion. Functional damage is inevitable.

With the chromosome- spindle complex far away from the first polar body, the inhalation method has a great effect on the physical shape of the nucleus oocytes in the case of inhalation or inhalation of the nucleus. There is a problem to increase the denuclearization of the chromosome- spindle complex.

Despite such a series of technical advances, fundamental development of egg denuclearization technology has not been achieved yet, and development of a denuclearization method capable of minimizing the aforementioned problems is required.

Even in the physical or chemical case as described above, since the nuclear transfer through the incision after denuclearization is the same process, the present invention can effectively physically denuclearize using the incision, nuclear transfer pipettes used for nuclear transfer at the same time. We present a new method for denuclearization.

The present inventors have endeavored to develop a novel denuclearization method that overcomes the above problems in the production of somatic cell embryos, and thus developed a double pipette consisting of an incision pipette and a nuclear transfer pipette. An incision was made in the nucleus oocyte with an incision pipette, and through this incision, the denuclearization rate and nucleation of the nucleus oocytes were simultaneously increased by the nuclear transfer pipette, thereby confirming that the production efficiency of the somatic cell cloning embryo was increased. .

Accordingly, the present invention is to provide a pipette used in the production of somatic replication fertilized egg, a double pipette consisting of an incision pipette and a denuclearization / nuclear transplant pipette, a nucleus nucleus denuclearization, nuclear transfer method and a somatic cloned animal production method using the same.

The present invention provides a double pipette 100 used in the production of somatic cell replication fertilized egg consisting of an incision pipette 10 and a denuclearization / nuclear transfer pipette 20.

More specifically, as a pipette used in the production of somatic cell replication fertilized egg consisting of an incision pipette 10 and a denuclearization / nuclear transfer pipette 20,

The incision pipette 10 has a constant inner diameter at one end and a capillary portion 11 having a shape that is open to the outside, and an inner diameter at the other end is shorter than an inner diameter of the capillary portion 11 and shorter toward the end. Consists of a closed tip shape 13 comprising a closed tip end 12 which is finely drawn and disconnected from the outside,

The denuclearization / nuclear transplant pipette 20 has a constant inner diameter at one end and a capillary portion 21 having an open outer portion, and an inner diameter at the other end thereof is shorter than the inner diameter of the capillary portion 21 and toward the end. It consists of an open tip shape 23 comprising an open tip end 22 that is open and externally finely pulled out to be shorter,

When the incision pipette 10 and the denuclearization / nuclear transplant pipette 20 are positioned in parallel in the same direction, the capillary portion 11 of the incision pipette 10 and the denuclearization / nuclear transplant pipette 20 The capillary portion 21 is coupled to each other, and when coupled in this way, the closed tip end 12 protrudes longer than the open tip end 22 to provide a double pipette (FIGS. 1 to 1). 3).

Such a double pipette 100 of the present invention can be used in the production of somatic cell replication fertilized eggs, in particular can be used for hydronucleated incision, denuclearization and donor nucleus transplantation.

The cutting pipette 10 and the denuclearization / nuclear transplant pipette 20 constituting the double pipette 100 of the present invention, first, using a micropipette puller (micropipette puller), the inner diameter is constant and both ends are open; The capillary can be manufactured and used in a form in which one end is finely drawn, which can be prepared using a conventional method of processing a capillary for micro injection in the art. At this time, the closed tip shape portion 13 and the open tip shape portion 23 of the incision pipette 10 and the denuclearization / nuclear transfer pipette 20 are pulled out to form a desired inner diameter and are formed. It is possible by adjusting conditions such as temperature, pulling force, etc. in the micro puller.

1 is a cross-sectional view of an incision pipette 10 for making an incision before denuclearization in a nucleolar egg. The incision pipette 10 is used as it is made of a puller. As one end, the capillary portion 11 has a constant inner diameter and a capillary shape open to the outside, and as the other end, the closed tip end 12 has an inner diameter. Shorter than the inner diameter of the capillary portion (11) is finely pulled so as to become shorter toward the end and is disconnected from the outside.

2 is a cross-sectional view of the denuclearization / nuclear transfer pipette 20 which denuclearizes through an incision in a nucleus pulmonary egg and implants a donor nucleus. As one end, the capillary portion 21 has a constant inner diameter and a capillary shape that is open to the outside, and as the other end, the open tip end 22 has a fine diameter such that the inner diameter is shorter than the inner diameter of the capillary portion 21 and shorter toward the end. It is pulled out and open to the outside. The denuclearization / nuclear transfer pipette 20 may be molded in a state in which the open tip end 22 is opened by grinding the closed tip end 12 of the incision pipette 10.

In addition, the open tip end 22 has a longer inner diameter than the nucleus so that the nucleus can move intact in the pipette when denuclearing or donor nucleus is implanted in a nucleus oocyte, and the inner diameter is about 10 to 30 micrometers. It is especially preferable that it is about 10-20 micrometers. If the inner diameter is less than 10 micrometers, it is not preferable because the nucleus may be damaged by the pipette inner wall when moving in the pipette, thereby lowering the development rate of the final somatic cloning embryo into the blastocyst.

The thus prepared dissection pipette 10 and the denuclearization / nuclear transplant pipette 20 are located in parallel in the same direction, and the capillary portion 11 and the denuclearization / nuclear transplant pipette 20 of the dissection pipette 10 are provided. Capillary portion 21 is coupled to each other is formed of a double pipette 100 of the present invention, when the closed tip end 12 is characterized in that protruding longer than the open tip end 22 (Fig. 3).

Adhesively fix the incision pipette 10 and the denuclearization / nuclear transfer pipette 20 using a common adhesive such as silicone, epoxy bond or glue gun to all or a part of the contact portion between the two capillary portions 11 and 21. Can be.

In addition, it is preferable that the closed tip end 12 protrudes compared to the open tip end 22 to perform the post-incision denuclearization and nucleation step, wherein the closed tip end 12 protrudes. The degree can be adjusted according to the size of the nucleus of the nucleus, about 100 ~ 1000㎛ is preferred for high denuclearization rate and blastocyst development rate, the shape of the protruding about 400 ~ 600㎛ double pipette of the present invention ( Most preferred).

In addition, in the double pipette 100 of the present invention, in the case of the denuclearization / nuclear transfer pipette 20, hydrogen fluoride (Hydrofluoric acid) 2 ~ in order to prevent the cells from sticking inside the denuclearization / nuclear transfer pipette 20. After washing three times, washing with distilled water is preferably coated with I-Gepal (SIGMA, I-3021, Octylphenyl-polyethylene glycol).

In addition, it is possible that both the incision pipette 10 is located above or below the operation of the nucleus nucleus, to manufacture the double pipette 100 of the present invention so that the incision pipette 10 is located above the micromanipulation It is easy and preferable in order to increase the rate of denucleation and blastocyst development.

The double pipette 100 of the present invention prepared as described above has the effect of shortening the working time by enabling the operation without de-nucleating in the nucleus ova and the operation of the donor nucleus without exchanging the pipette in one working drop.

In addition, the denuclearization and nuclear transfer of the double pipette (100) of the present invention to the egg through the incision through the incision of the nucleus-nucleated egg (3), which is impossible to denuclear because the chromosome-church complex (8) and the first polar body (6) of the egg are not close to each other. Simultaneous denuclearization and nuclear transfer with the pipette 20 effectively removes the chromosome-chimney complex 8 far from the first pole 6 to increase the denuclearization rate.

In addition, the present invention provides a method for denuclearizing and nucleating the nucleus of the nucleus by using the double pipette 100 of the present invention in the production of somatic cell replication fertilized eggs.

Nucleated oocyte denuclearization and nuclear transfer method of the present invention, 1) making an incision in the nucleus egg (3) with a pipette for cutting 10 of the double pipette (100); 2) denuclearizing the denuclearization / nuclear transfer pipette 20 of the double pipette 100 through the incision; 3) The denuclearization and nuclear transfer pipette 20 of the double pipette (100) consists of the step of performing a donor nucleus at the same time.

The use of the double pipette of the present invention in the production of somatic cell replication embryos allows denuclearization and nuclear transfer at the same time, thereby reducing work time, enabling denuclearization while maintaining the morphology of the nucleus of the nucleus, and the nucleus of the nucleus of the nucleus oocytes. Efficient denuclearization and nuclear transfer are possible even when the plates are not in close proximity.

In addition, by increasing the denuclearization rate of the nucleus egg nucleus, it is possible to further secure the number of nucleus egg embryos provided in the production of cloned fertilized eggs to increase the development rate of blastocysts of cloned fertilized eggs after denuclearization.

Therefore, the double pipette of the present invention and the denuclearization method of the nucleophile egg using the same can be usefully used for the production of somatic clones.

The present invention provides a method for producing a somatic cell clone, including a nucleus ova denuclearization and nuclear transfer method using the double pipette (100).

1) culturing somatic cells and separating them into single cells to prepare donor nuclear somatic cells; 2) in vitro maturing the oocytes collected for nucleated oocytes; 3) removing the cumulus cells from the matured egg; 4) making an incision window using the incision pipette 10 of the double pipette 100 in the egg of step 3); 5) preparing a nucleus oocyte 3 by denuclearizing the denuclearization / nuclear transfer pipette 20 of the double pipette 100 through the incision of the egg; 6) preparing a donor nuclear transfer embryo by transplanting a donor nucleus (9) into the denuclearization / nuclear transfer pipette (20) of the double pipette (100) in the nucleus nucleus (3); 7) fusion of the donor nuclear transfer embryos to produce somatic replication embryos; 8) activating the somatic replication embryo; 9) ex vivo culture of somatic cloned fertilized egg; and 9) producing a somatic cloned animal by transplanting the somatic cloned fertilized egg into surrogate mother animals.

As used herein, 'somatic cells' refers to cells other than germ cells.

The 'donor nucleus somatic cell' of the present invention refers to a cell that provides genetic material in the nucleus as a nucleophile egg during nuclear transfer of somatic cells.

The term "nucleated oocyte" of the present invention refers to an egg which receives a nucleus from a donor nucleus somatic cell during nuclear transplantation of a somatic cell, and in which an nucleus in an egg is removed and provides a cytoplasm for nuclear transplantation of a donor nucleus somatic cell.

'Somatic cell replication fertilized egg' of the present invention refers to an egg in which the nucleus and the cytoplasm are fused by a physical or chemical method after injecting the donor nucleus of the somatic cell into the nucleus-nucleated egg.

'Somatic cells' of the present invention are animal cells, including, but not limited to, fish, amphibians, birds and mammals. Preferably somatic cells are mammals such as primates including humans, cattle, pigs, bears, cats, dogs, horses and rodents, and most preferably bovine or human somatic cells.

In the present invention, a basic method for producing somatic cloned embryos, ex vivo culture and transplantation into surrogate mothers to produce somatic cloned animals is in accordance with conventional methods known in the art.

In addition, the present inventor has disclosed a method for producing a somatic cloned animal in Korean Patent Publication No. 2002-0080616, the disclosure of which is incorporated herein by reference in its entirety and is not described herein. The features of are explained more clearly.

This increases the denuclearization rate of nucleated eggs by the present invention in the case of difficult to secure nucleated oocytes, thereby enabling to more secure the number of somatic cell embryos developed by nucleated oocytes and blastocysts during the production of cloned eggs. As a result, the production rate of somatic cloned animals is also increased.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

[ Example 1 Double pipette preparation of the present invention

In order to prepare the double pipette of the present invention, a pipette for dissection and denuclearization and nuclear transfer was prepared, respectively.

Micro pipettes were manufactured using devices such as Puller (PC-10, Narishigae, Japan), Microforge (MF-9, Narishige, Japan) and the like.

First, the cutting pipette (Cutting pipette) is attached to the weight (1 small, 1 large) using G-1 (outer diameter 1.0mm, inner diameter 0.9mm, Narishige, Japan), and pulling step (step) After fixing to 1 ~ step 2), it was prepared by adjusting the temperature to 80 ~ 101.5 ℃.

Injection pipette (Dejection pipette) is attached to the weight (1 small) using G-1, and fixed in the pulling step (step 1 ~ step 2), and then the temperature is adjusted to 80 ~ 101.5 ℃. It was prepared by. The pipette for denuclearization / nuclear transplantation was washed 2-3 times with hydrogen fluoride (Hydrofluoric acid), and then with distilled water and coated with I-Gepal (SIGMA, I-3021) to prevent cells from sticking inside.

Then, the incision pipette 10 and the denuclearization / nuclear transplant pipette 20 are placed in parallel, and the closed tip end 12 of the incision pipette is the open tip end of the denuclearization / nuclear transplant pipette 20 ( It is positioned to protrude about 500 ㎛ compared to 22) so that it does not interfere with each other during incision and nuclear implantation, and then it is adhered to the part where the capillary portions (11, 21) of both pipettes are contacted with silicon, combined and fixed integrally. A double pipette of the present invention was prepared (see FIGS. 1-4).

[ Example 2 Production of Somatic Replication Embryos and Somatic Cloning Animals Using the Double Pipette of the Present Invention

1) Collection and Cultivation of Oocytes

From the ovaries of cattle collected from the slaughterhouse, only follicles with a diameter of 3 to 5 mm were selected, and immature follicles with uniform cytoplasm and follicular cells under a stereoscopic microscope were obtained after extracting eggs with follicular fluid using an 18-gauge needle and a disposable syringe. Screened. Immature follicles were incubated in incubator adjusted to 5% CO ₂ , 95% humidity for 20-20 hours using mature culture medium. As the maturation culture medium, TCM199 (hereinafter referred to as TCM199) added with 10% FBS, 0.015 IU FSH (Antrin, Denka Pharm., Japan), and 0.015 IU LH (Sigma, USA) was used.

2) somatic cell culture

2-1) Donor nucleus  Somatic cell isolation

The 40-day-old fetus was chopped with ophthalmic scissors, and excised into 0.1-0.2 cm 2 wide tissues. Then, the phosphate buffer solution was prepared in a 5 ml test tube, and the refrigerated tissues were put in a refrigerator at 4 ° C. After washing with phosphate buffer solution, 0.05% trypsin and 1 mM EDTA solution were added and allowed to stand for 10-20 minutes in the incubator. After centrifugation for 5 minutes at 1800rpm it was washed 2-3 times. After final centrifugation, the precipitated cells were transferred to DMEM with 10% FBS and cultured in a 37 ° C., 5% CO ₂ saturated humidity incubator.

2-2) Subculture

The petri dishes were discarded and washed 1 to 2 times with phosphate buffer, followed by addition of 0.05% trypsin and 1 mM EDTA solution. The cells were washed with phosphate buffer, recovered, centrifuged and washed at 1800 rpm for about 5 minutes, then aliquoted into a new petri dish and cultured in a saturated humidity incubator at 37 ° C. and 5% CO ₂ .

2-3) Serum starvation  culture

The proliferating cell lines were cultured in DMEM added with 10% FBS and replaced with a culture solution added with 0.5% FBS, and provided as donor nuclei after 3 to 10 days of culture.

3) physical Denuclearization

3-1) Removing Oocytes

All in vitro manipulations were performed using mSOF (hereinafter, Hepes-mSOF) with 25 mM Hepes added. Culture conditions are 5% CO ₂ , 5% O ₂ , high humidity environment, the composition of the in vitro culture (NaCl (106mM), KCL (7.2mM), NaHCO ₃ (25mM), KH ₂ PO ₄ (1.2mM), sodium Lactate (6.6 mM), CaCl ₂ (1.7 mM), MgCl ₂ (0.5 mM), sodium pyruvate (0.3 mM), glucose (1.5 mM), BSA (8 mg / ml), MEM essential amino acid solution (2% v / v), MEM non-essential amino acid solution (1% v / v), glutamine (1 mM), ITS insulin, transferrin, selenium mixture (1%, v / v).

The oocytes matured for 20 hours were washed 3-4 times with Hepes-mSOF to which 0.1% hyaluronidase (Sigma, USA) was added, followed by gentle pipetting using a glass tube having an inner diameter of 170 μm. The remaining cumulus cell layer was removed. In the experiment, only mature eggs with homogeneous cytoplasm and the first polar body were released were used. This work time was within 10 to 15 minutes.

3-2) double pipette of the present invention In nuclear transfer  Create a pipette to use

As a pipette to be used for nuclear transfer, a pipette for fixation, a double pipette of the present invention, and a pipette for washing and transporting eggs were prepared, respectively.

Pipettes were manufactured using devices such as Puller (PC-10, Narishigae, Japan), Microforge (MF-9, Narishige, Japan) and the like.

The holding pipette (50) is fixed to the pulling step (Pulling step, step 1) without attaching weight using GD-1 (Narishige, Japan), and then adjusts the temperature to 80 ° C. Prepared.

The double pipette of the present invention was prepared by integrating the two pipettes integrally by making the incision pipette and the denuclearization / nuclear transfer pipette as in Example 1 (FIGS. 1 to 4).

The pipette for washing and transporting the eggs was made to have an inner diameter of 300 µm or more using an alcohol lamp, and used one was discarded.

The produced pipettes were placed in a pipette fixing device of the manipulator for micromanipulation of fertilized eggs. The following operation was performed by alternately moving the double pipette of the present invention to a desired position using a driving device provided in the manipulator.

3-3) Transparent  Incision and Denuclearization

The mature egg is classified into an egg to be denuclearized using the double pipette of the present invention (experimental group), an egg to be denuclearized using a conventional squeeze method (comparative group 1) and an egg to be denuclearized with an oocyte fixation pipette (comparative group 2). As shown, a zona pellucida incision and denucleation were performed.

First, the nucleus nucleus was allowed to stand for 10-15 minutes in a mixed solution of Hoechst 33342 (final concentration 5-10 µg / ml) and cytocalasin B (cytochalasin B, sigma C-6762, final concentration 5 µg / ml). This was transferred to 4 μl of Hepes-mSOF microdrops containing 100 μg / ml Phytohematoglutinin (Shyma, U.S.A.), followed by denuclearization.

The egg of the experimental group was fixed to the first polar body in the 12 o'clock direction with a fixing pipette, and then the incision pipette of the double pipette of the present invention in the 1 o'clock direction, and the incision was made at the closed tip end in the 10 o'clock direction. Passed. Thereafter, after the fixing pipette was loosened, the fixing pipette was contacted with the transparent zone of the upper end of the first polar body passing through the incision pipette, and the two pipettes were rubbed to make an incision in the transparent zone. Then, in the egg of the experimental group, a portion of the cytoplasm containing the first polar body and the medium substrate was removed from the egg by applying a negative pressure using a nuclear transfer pipette of the double pipette of the present invention (Figs. 5 and 6).

Comparative group 1 is a group subjected to denuclearization by squeegeeing method, and fix the egg so that the first polar body goes to the 12 o'clock position with a fixing pipette, and then cut the transparent band with the incision pipette at 1 o'clock, Passed through. Thereafter, after the fixing pipette was loosened, the fixing pipette was contacted with the transparent zone of the upper end of the first polar body passing through the incision pipette, and the two pipettes were rubbed to make an incision in the transparent zone. Then, in the egg of Comparative Group 1, a part of the cytoplasm containing the first polar body and the medium substrate was released from the egg under pressure (FIG. 7).

On the other hand, the egg of the comparative group 2 makes an incision in the same way as the egg of the experimental group, and gives a negative pressure through the incision using an oocyte fixation pipette, and in the egg releases a part of the cytoplasm containing the first polar body and the medium substrate from the egg (FIG. 8).

4) Nuclear transfer

As in the denuclearization process, nucleation was performed in the same working drop of 4 μl Hepes-mSOF containing 100 μg / ml phytohematoglutinin.

After a small amount of the somatic cell suspension, which is the donor nucleus prepared in Example 2-2, was injected into the working drop in the state of denuclearization, clean cells of 20 µm in diameter were selected and sucked into the nuclear transfer pipette. Then, after fixing the egg so that the incision of the denucleated egg is in the 1 o'clock direction with a fixing pipette, the nuclear transfer pipette is transferred into the ostral cavity through the incision and the somatic cells are injected.

When the nuclear transfer after denuclearization using the pipette for nuclear transfer of the double pipette of the present invention, as in the experimental group, it becomes the shape as shown in FIG. 9.

When performing the denuclearization and nuclear transfer process using the double pipette of the present invention, it is possible to carry out nuclear transfer at the same time with the denuclearization to reduce the working time, to enable the denuclearization while maintaining the form of the nucleus of the nucleus, the nucleus of the nucleus 1 Efficient denuclearization and nuclear transfer were possible even when the pole and the mid board were not in close proximity.

When nuclear transfer is performed after denuclearization in Comparative Group 1 and Comparative Group 2, the denuclearized oocyte first pole and the medium substrate are shown in the incision window as shown in FIG. (FIG. 7). When the chromosome- spindle was not close in Comparative Group 1 and Comparative Group 2, there was a problem in that the chromosome- spindle complex was denuclearized by increasing the amount of cytoplasm leaked to the outside.

5) Electric fusion

After nuclear transfer was completed, the cytoplasm of donor nucleus (9) cells and nucleated oocytes (3) was fused with Electronic Cell Manipulator RMX2010 (www.Biofusiontech.com, Korea) (FIG. 10). In this case, 0.27 M mannitol (Sigma, United States) solution to which 0.1 mM MgCl ₂ and 0.05% BSA was added was used, and the cells were allowed to equilibrate by leaving the nuclear transplanted eggs for 2-3 minutes. Next, the nuclear transfer cell is placed directly between the needles for electrofusion, and the donor nucleus (9) cells are directed to the (+) pole and the egg to the (-) pole, and the direct fusion needle is applied with a slight positive pressure to the surface of the nucleus egg. 20 Volt, time applied 10 ㎲, number of times was stimulated to induce fusion. After 30 minutes, the fusion was confirmed and the fusion was not performed once.

6) Induce reprogramming

After completion of the electrofusion replication clones were incubated for 4 hours in mSOF for 4 hours at 39 ℃, 5% CO ₂ incubator to induce the reprogramming of the nucleus.

7) chemical activation

The nuclear transfer embryos were transferred to mSOF for culture, incubated for 30 minutes, and then selected and activated only for eggs that had normal fusion.

Ca ^{2 +} -Ionophore (A23187) that were activated with the egg activate for 5 minutes in Hepes-mSOF solution 6-DMAP is added in addition mSOF solution for 4 hours. The activated eggs were washed in mSOF medium 2-3 times and cultured.

8) Culture of cloned fertilized eggs

After activating the cloned embryos were washed three times with Hepes-mSOF and then cultured and differentiated at 39 ° C., 5% CO ₂ incubator in mSOF for 7 days to develop blastocysts (FIG. 12, FIG. 13).

The blastocyst development rate of each of the cloned eggs prepared in Examples 1) to 8) is shown in Table 1 (results of the cloned eggs of Example 3-3) and Table 2.

Number of eggs Number of developed somatic cloning embryos (%) Denuclearization Number of eggs before denuclearization Number of denuclearization successes (A) Injection Fusion (B) 2 cells (C) 4-cell (C) 8 cells (C) 16 cells (C) Lost Belly (C) Blastocyst (C) Experimental group Use of double pipettes of the present invention 172 146 (85%) 141 132 (94%) 101 (84%) 95 (72%) 71 (54%) 46 (35%) 40 (30%) 33 (25%) Comparative Group 1 Squeezing 156 109 (70%) 103 98 (95%) 76 (78%) 66 (67%) 56 (57%) 39 (40%) 26 (26%) 20 (20%) Comparative group 2 Fixed pipettes simultaneously 138 95 (69%) 92 85 (92%) 66 (77%) 55 (65%) 46 (54%) 27 (32%) 24 (28%) 19 (20%) A percentage of the number of pre-nucleated eggs B percentage of the number of injected eggs C percentage of the number of fused eggs

In the results of Table 1, the denucleation success rate was the highest in the experimental group denuclearized using the double pipette of the present invention as 85%, and the blastocyst development rate of cloned fertilized eggs produced by nuclear transplantation afterward was the highest as 25%. Could.

The development of cloned fertilized blastocysts in Comparative Group 2, which had undergone nuclear transfer after denuclearization with a fixed pipette, had a high development rate of 20%, and the development rate of cloned fertilized blastocysts in Comparative Group 1 prepared by squeezing method and each method had a significant difference. I didn't see it.

In addition, the ratio of the total number of donated eggs referred to as an important point in the present invention

Denuclearization Number of eggs before denuclearization Denuclearization success number Blastocyst Experimental group Use of double pipettes of the present invention 172 146 (85%) 33 (19.1%) ^D Comparative Group 1 Squeezing 156 109 (70%) 20 (12.8%) Comparative Group 2 Use of fixed pipettes 138 95 (69%) 19 (13.7%) D Percentage of number of prenucleated eggs

Looking at the percentage of the total number of eggs, using the double pipette of the present invention it was confirmed that the development rate of the blastocyst for the total number of eggs of the cloned fertilized eggs (experimental group) prepared by denuclearization, nuclear transplantation was the highest (19.1%).

On the other hand, the development rate of blastocysts (comparative group 1) produced by squeezing method was 12.8%, and the development rate of blastocysts (compared group 2) after denuclearization with fixative pipette was 13.7%, respectively.

In other words, the squeegeeing method (Comparative Group 1) and the denuclearization method using the fixed pipette (Comparative Group 2) did not show a significant difference in the blastocyst percentage to the total number of eggs before denuclearization, but the denuclearization was performed using the double pipette of the present invention. The method (experimental group) had a high rate of denucleation and a high rate of development of blastocysts, which showed a significant difference in the final result.

In the case of Comparative Group 1 and Comparative Group 2, when the chromosome- spindle is not close, there is a problem of denuclearizing the chromosome- spindle complex by increasing the amount of cytoplasm leaked to the outside, and Comparative Group 1 and Comparative Group 2 are relatively After denuclearization, cytoplasmic loss increases, resulting in poor adhesion between the donor nucleus and the cytoplasm even when donor nuclei are fused, resulting in a decrease in cell fusion rate and the development rate of blastocysts.

Therefore, the double pipette consisting of the incision pipette and the nuclear transfer pipette of the present invention and the denuclearization method of the nucleus-nucleated egg using the same are provided during the production of cloned fertilized eggs by increasing the denuclearization rate of the nucleus-nucleus egg in a mammal that is difficult to secure the nucleus-nucleus egg. It is possible to further secure the number of the nucleus of the nucleus, which has the effect of increasing the blastocyst development rate of the cloned fertilized egg after denucleation.

9) Transplantation of somatic cloned embryos and production of somatic cloned animals

The results of producing somatic cloned animals by transplanting somatic cloned fertilized eggs of the in vitro cultured experimental group into surrogate mothers (Holstein cows) according to a conventional fertilized egg transplant method are shown in Table 3 below.

Blastocyst maturation eggs Transplant head During pregnancy Birthman 30 6 3 One

As described above, the use of the double pipette of the present invention in the production of somatic cell replication fertilization enables nuclear transplantation at the same time with denuclearization, reducing the working time, enabling denuclearization while maintaining the morphology of the nucleus ovary, Efficient denuclearization and nuclear transfer are possible even when the nucleus of the nucleus of the nucleus is not close.

In addition, by increasing the denuclearization rate of the nucleus oocytes, it is possible to further secure the number of nucleus oocytes provided in the production of cloned fertilized eggs, thereby increasing the development rate of blastocysts of cloned fertilized eggs after denuclearization.

Therefore, the denuclearization and nuclear transfer double pipette of the present invention and the nucleation and nuclear transfer method of the nucleus of the nucleus using the same can be usefully used for the production of somatic clones.

Claims (2)

A method of producing a somatic cloned animal using a somatic cloned fertilized egg prepared by denuclearizing and nucleating a nucleated oocyte using a double pipette consisting of an incision pipette and a nucleus / nucleus transfer pipette in the manufacture of a somatic cloned animal. The denuclearization / nuclear transplant method comprises the steps of: 1) making an incision in the nucleus ovum with the incision pipette of the double pipette; 2) denuclearizing the nuclear pipette of the double pipette through the incision; And 3) simultaneously performing donor nuclear transfer with the denuclearization / nuclear transfer pipette of the double pipette; The incision pipette is a capillary portion of a capillary shape having a constant inner diameter and open to the outside as one end thereof, and a closed type that is pulled out so that the inner diameter is shorter than the inner diameter of the capillary portion and shorter toward the end as the other end. A closed tip shape comprising a tip end, The denuclearization / nuclear transplant pipette has a capillary portion of a capillary shape having a constant inner diameter and open to the outside as one end thereof, and an outer diameter that is pulled out so that the inner diameter is shorter than the inner diameter of the capillary portion and shorter toward the end as the other end thereof. An open tip shape comprising an open tip end, When the incision pipette and the denuclearization / nuclear transfer pipette are positioned in parallel in the same direction, the capillary portion of the incision pipette and the capillary portion of the denuclearization / nuclear transfer pipette are coupled to each other, and thus closed Method of producing a somatic cell clone using a double pipette, characterized in that the tip end is protruded longer than the open tip end. The method of claim 1, further comprising the steps of: 1) culturing somatic cells and separating them into single cells to prepare donor nucleus somatic cells; 2) in vitro maturing the oocytes collected for nucleated oocytes; 3) removing the cumulus cells from the matured egg; 4) making an incision using the incision pipette of the double pipette in the egg of step 3); 5) preparing a nucleated egg by denuclearizing the denuclearization / nuclear transfer pipette of the double pipette through the incision of the egg; 6) preparing a donor nuclear transfer embryo by transplanting a donor nucleus into a denuclearization / nuclear transplantation pipette of a double pipette into the nucleus nucleus oocyte; 7) fusion of the donor nuclear transfer embryos to produce somatic replication embryos; 8) activating the somatic replication embryo; 9) ex vivo culture of somatic cloned fertilized egg and 9) transplanting the somatic cloned fertilized egg into a surrogate mother animal to produce somatic cloned animal.

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