KR20040079189A - Method for production of cloned animal embryos by nuclear transfer - Google Patents

Abstract

PURPOSE: A method for production of cloned animal embryos by nuclear transfer is provided to reduce the number of cloning processes. Therefore, itt can be useful for preservation of animals threatened with extinction, production of useful proteins, development of disease model, and supply of deficient organs. CONSTITUTION: The method for production of cloned animal embryos by nuclear transfer comprises the steps of: (i) inhaling a first polar body and 10 to 20% of cytoplasm without dying the nuclear of an egg cell by using a glass pipe having diameter of 20 to 40 micrometer to remove the nuclear from the egg cell; (ii) introducing a somatic cell into the first polar body and nuclear removed egg cell to prepare an egg-somatic cell complex within 1 minute during the inhalation process; and (iii) subjecting the egg-somatic cell complex to electric stimulation of 0.5 to 2 kv/cm and 6 to 150 microseconds 1 to 3 times to fuse and activate the egg-somatic cell complex, wherein the egg cell is collected from a mammal selected from pig, cow, sheet, mouse, rat, rabbit, dog and cat.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a method for producing a cloned animal embryo by nuclear transfer,

The present invention relates to a method for producing an animal clone by nuclear transfer, and more particularly, to a method for producing an animal clone by nuclear transfer, comprising the steps of removing the first polar body and nucleus of an oocyte and injecting somatic cells into the egg- To a method for producing a clone.

Cloning is genetically identical, and nuclear transfer is a new method of replication in eukaryotes that has recently come to the fore with the rapid development of molecular biology. Early nuclear transfer has been used as a source of nuclear fertilized eggs (Prather et al ., Biol. Reprod. , 1987, 37: 856-866; Prather et al ., Biol. Reprod. , 1989, 414-418). Recently, somatic cell nuclei have been used.

Somatic replication is a technique in which differentiated somatic cells are activated by putting them into a nucleus-free oocyte and then generated in the same way as normal embryos. Such replication technology is expected to be widely used for research in basic science including developmental biology, and is expected to contribute greatly to various medical and pharmaceutical fields such as production of useful protein, development of disease model, and organ transplantation. It can be said that the usability is very high.

Dolly, the first animal cloning. (Willmut, I. et al, Nature, 1997, 385:. 810-813) after the amount of birth, cloning cows in the field continued to develop many (Cibelli, JB et al. , Science, 1998, 280: 1256-1258 ; Wells, DN et al, Reprod Fertil Dev, 1998, 10:...... 369-378), mice (Wakayama, T. et al, Nature , 1998, 394 : 369-374), goat (Bagusi, A. et al ., Nat. Biotechnol ., 1999, 17: 456-461). However, pigs have had relatively few studies on fertilized eggs compared to other species, and at least four embryos have to be implanted in the uterus to survive cloning, was (Poleajaeva, IA et al, Nature , 2000, 407: 86-90; Onishi, A. et al, Science, 2000, 289:..... 1188-1190; Betthauser, J. et al, Nat Biotechnol. , 2000, 18: 1055-1059). After that, the first transgenic cloned pig was born by transplanting an exogenous gene into pig somatic cells and succeeded in cloning (Park, KW et al ., Anim. Biotechnol ., 2001, 12: 173-181) Reproduction piglets that have knocked out somatic cells have also been successful (Lai, L. et al., Nat. Biotechnol. , 2002 Mar. 20 (3): 251-255). However, despite the rapid development of pig somatic cell replication, replication efficiency is still low at 1-5%, and many studies are under way to improve efficiency.

The key technology in somatic cell replication technology is the process of removing the nucleus of the egg prior to injecting somatic cells into the stomach of the oocyte during nuclear transfer. Fluorescent staining, hoechst 33342, is commonly used to remove the nucleus of the oocyte. That is, oocytes are stained with Hest 33342, and the oocyte nuclei are removed under ultraviolet light (UV). At this time, the fluorescent staining or exposure to ultraviolet light is likely to affect the development of nuclear transfer embryos (Tao, T. et al ., Zygote , 2000, 8: 69-77) Causing a great deal of fatigue, thereby decreasing the production efficiency of the cloned cells.

Disclosure of the Invention The present invention has been conceived to solve the above problems. It is an object of the present invention to simplify the nuclear transfer process by removing the nucleus without using fluorescent staining and ultraviolet rays and activating the cloning column without additional activation stimulation after oocyte and somatic cell fusion The present invention also provides a method for cloning somatic cells with high efficiency.

1 is a photograph showing a method of removing nuclei from an egg without going through fluorescent staining,

Ⓐ Fix the polar body of egg to 1 o'clock position,

Ⓑ About 10-20% removal of cytoplasm around the polar body,

Ⓒ Remove the nucleus and insert somatic cells into the urean steel,

Ⓓ Somatic cells are inserted in the stomach

Fig. 2 is a photograph showing the nucleus stained after the cloning column was cultured for 6 days.

Ⓐ general microscope photograph,

Ⓑ fluorescence microscope photograph after fluorescence staining

In order to accomplish the above object, the present invention provides a method for removing oocytes, comprising the steps of: i) removing the nucleus of an oocyte by suctioning the first polar body and 10-20% of the cytoplasm without staining the nucleus of the oocyte; Ii) injecting a somatic cell into the proliferation of the oocyte in which the first polar body and the nucleus are removed to prepare an oocyte-somatic cell complex; And iii) cell fusion and activation of the oocyte-somatic cell complex by electrical stimulation conducted at 0.5 to 2 kV / cm and 6 to 150 1 for 1 to 3 times.

The term "polar body" refers to a protrusion of a part of ovarian cells separated by an oocyte by the second meiosis before fertilization (see Fig. 1), and the "Yurian River " refers to a space between the oocyte's zona pellucida and the cytoplasm. In addition, "oocyte-somatic cell complex" refers to a stage in which somatic cells are injected into the rabbits after oocyte nuclei are removed, before the cell fusion occurs, and "cloned cells " refers to a stage where oocytes and somatic cells are fused by electrical stimulation.

Hereinafter, the present invention will be described in detail.

The replication method of the present invention comprises the steps of: i) removing the nucleus of the oocyte by inhaling the first polar body and 10-20% of the cytoplasm without staining the nucleus of the oocyte; Ii) injecting a somatic cell into the proliferation of the oocyte in which the first polar body and the nucleus are removed to prepare an oocyte-somatic cell complex; And iii) 0.5 to 2 kV / cm, 6 to 150 1 for 1 to 3 times to effect cell fusion and activation of the oocyte-somatic cell complex.

It is preferable that the inhalation of step i) is carried out using a hydraulic pressure difference using a glass tube having a diameter of 20-40 mu m and a tip structure of 30-60 DEG.

Specifically, as in the embodiment of the present invention, the oocyte's polar body is first fixed at 1 o'clock position, and the micro-glass tube sharpened at 30-60 DEG is pushed in the polar body direction. By using the hydraulic pressure, the cytoplasm around the polar body and the polar body is inhaled by about 10 to 20%. At this time, since most of the nuclei are located around the polar body, most of the nuclei of the oocyte are removed in the above process.

In addition, in step i), the egg for producing the cloned egg is preferably collected from a mammal other than a human, and is preferably composed of a pig, a cow, a goat, a mouse, a rat, a rabbit, And more preferably selected from the group consisting of The somatic cells to be cloned are preferably collected from mammals other than humans, and more preferably selected from the group consisting of pigs, cows, goats, mice, mice, rabbits, dogs and cats, Most preferred.

In addition, in step ii), it is preferable to inject somatic cells within one minute after removing the nucleus of the oocyte.

In the prior art, since the nuclei of all the oocytes to be used for nuclear transfer are removed and then the somatic cells are injected, the time required for removing the nucleus of the oocyte takes about 1-3 hours before the somatic cells are injected. However, since the somatic cell implantation process is performed immediately after the removal of the nucleus of the oocyte, the time can be shortened to one minute.

In addition, in step iii), it is preferable that the electric stimulation is performed twice at 1.0 to 1.2 kV / cm and 30 μs.

In this step, the somatic cells injected into the proliferation of the oocyte-free oocyte are fused with an electric stimulus (Poleajaeva, IA. Et al ., Nature , 2000, 407: , 2002, 12: 173-181). After fusion, the electrical stimulation was observed in the presence of an electrical stimulus (see, for example, Beththauser, J. et al ., Nat. Biotechnol ., 2000, 18: 1055-1059; Park, KW et al ., Anim. Biotechnol . Or chemical stimulation to induce activation of the cloned cells.

The replication method of the present invention not only simplifies the replication process by simplifying the nuclear transfer process of somatic cells, but also promotes its production efficiency.

First, when removing the nucleus of the oocyte, a DNA specific fluorescent material for fluorescent dye generally used conventionally is not used, and the nucleation rate is also high by the above method. In general, when removing the nucleus of an egg, conventionally, a DNA-specific fluorescent dye material has been widely used. However, a fluorescent chromosome stained with DNA can adversely affect the oocyte (Tao, T. et al., Zygote , 2000, 8: 69-77), and ultraviolet light irradiated to examine the fluorescent chromosome It is known that it can affect. In the present invention, the nucleus is efficiently removed from the egg while omitting the above process.

Second, somatic cells are injected immediately after oocyte nucleus is removed. Conventionally, the nuclei of whole oocytes were first removed one by one to remove the nuclei of all the oocytes, and somatic cells were injected. The nucleus method requires that the pores generated in the nucleus of the oocyte are recomputed when the somatic cells are injected. If a new hole is made in the oocyte for the somatic cell implantation, the cytoplasmic component of the oocyte may be leaked through the hole formed in the nucleus , Somatic cells were injected through the pores produced during the nucleus. At this time, it is not easy to find holes in the nucleus in the oocyte, and it takes considerable time, and also gives considerable stress to the clone. Therefore, in the case of producing more than 100 replicates, the method of injecting somatic cells immediately after the nucleus as in the present invention alleviates the inconvenience described above and can save a considerable amount of time.

Third, it induces cell fusion and activation by a single electric shock. In nuclear transfer embryos, oocytes and somatic cells are generally cell fusion by electrical stimulation (Poleajaeva, IA. Et al ., Nature , 2000, 407: 86-90; Betthauser, J. et al ., Nat. Biotechnol ., 2000, 18 : 1055-1059; Park, KW et al ., Anim. Biotechnol ., 2001, 12: 173-181). After a lapse of a certain period of time after the cell fusion, activation of the replicase is induced through another electrical stimulation or chemical stimulation (Poleajaeva, IA. Et al ., Nature , 2000, 407: 86-90; Betthauser, J. et al . et al, Nat Biotechnol, 2000, 18:... 1055-1059), conventionally, the number of cells increased after the blastocyst ( "embryos are divided several times in the case of inducing additional active after such fusion cells to form and contains baegang (Pre-implantation in the uterus) is generally 7 to 10% (Betthauser, J. et al ., Nat. Biotechnol ., 2000, 18: 1055-1059; Koo, DB. Et al , & Lt; / RTI > Biol Reprod ., 2000, 63: 986-992). However, in the present invention, the incidence rate to the blastocyst is as high as 14.6 to 16.6% even if no stimulation is given (see Fig . 1 ). In the present invention, as in the prior art, the incidence rate to the blastocyst by the additional electrical stimulation of 1 to 2 hours after fusion did not show a large difference from the incidence.

This simplification of the replication process has the advantage of minimizing stimulation on the oocyte and the replication field by not only minimizing the in vitro manipulation time of the oocyte and the cloning site, but also eliminating the additional stimulation for activation of the fluorescence staining and replication field. In addition, with the minimization of the damage to the replication field, there is an additional effect of minimizing the reduction of the manufacturing efficiency of the replication field due to fatigue of the manufacturer by reducing the working hours of the manufacturer of replication.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1: Collection of oocytes

The ovaries of ginseng were collected from slaughterhouses and stored at 35 to 39 ° C in 0.9% physiological saline. An 18-gauge needle was connected to a 10-ml syringe and follicular fluid was inhaled in the ovarian follicles 2 to 6 mm in diameter to collect eggs. The oocytes were washed in a mature culture medium, and 50 to 60 oocytes were added to 500 쨉 l culture medium and cultured for 42 to 46 hours.

At this time, the mature culture solution was added to 0.1% polyvinyalcohol, 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, (Sigma), 0.5 g / ml FSH (Sigma), 10 ng / ml epidermal growth factor (Sigma), 75 占 퐂 / ml penicillin G and 50 [mu] g / ml streptomycin.

Example 2: Culture of somatic cells

The pregnant pigs were slaughtered between 30 and 35 days of gestation and the pig embryos were picked and placed on ice for transportation to the laboratory. Porcine embryos were cut with scissors and pig somatic cells were cultured in PBS (phosphate buffered saline) supplemented with 0.05% trypsin and 0.5 mM EDTA for 30 minutes. The supernatant was removed by centrifugation and then cultured in DMEM (Dulbecco's modifed Eagle medium) containing 10% fetal calf serum (BioWhittaker) for 2-3 days. After 80% confluency of the bottom (80% confluency), the cells were subcultured by trypsinization, and some of the cells were used for nuclear transfer and the remaining cells were cryopreserved.

≪ Example 3 > Micro-manipulation (micromanipulation)

The oocytes collected in Example 1 were cultured for 5 to 10 minutes in the micronutrient broth, and the somatic cells cultured in Example 2 were added to the culture broth. At this time, the microorganism culture broth was prepared by adding 0.3% BSA and 7.5 / / ml CB (cytochalasin B) to TCM 199.

To remove the oocyte nuclei, a micro-glass tube having a diameter of 30 탆 and sharpened at a tip of 30-60 ° was prepared. First, the oocyte's polar body was fixed at 1 o'clock, and the micro-glass tube was stuck in the polar body direction. Most of the oocyte nuclei were removed in the process because most of the nuclei were located around the polar body, using the hydraulic pressure to inhale the cytoplasm around the polar body and the polar body. Thereafter was sucks the porcine somatic cells prepared in Example 2 using the fine glass tube was charged with the somatic cell by using a hydraulic After placing the wiran steel of the oocyte nucleus is removed, the fine glass tube to wiran steel of the egg (Fig. 1 ).

Example 4 Dyeing of oocyte nuclei

In order to confirm the degree of the nucleus of the oocyte after the above Example 3, some oocytes were subjected to fluorescence staining with Hest 33342 (Sigma) after eliminating the nuclei without injecting somatic cells ( FIG. 2 ) .

This is different from fluorescence staining prior to the nucleus in the prior art. In the present invention, after completion of the nucleus, fluorescence staining was performed to discriminate the degree of the nucleus. As a result, it was confirmed that the nucleation rate was 86.3% in 350 repetitions, so that it was possible to remove the nucleus from the oocyte with high efficiency without using fluorescence staining before the nucleus by using the method of the present invention. Thus, in the following example, the steps of Example 5 were followed immediately after the nucleotide of Example 3 without any fluorescent staining.

≪ Example 5 > Culture of cell fusion, activation and replication

<5-1> Cell fusion and activation

When the micro-manipulation of Example 3 was completed, the oocyte-somatic cell complex was placed between the platinum wire lines spaced 1 mm apart after inserting the nuclear transfer embryos into the activation culture medium, and a 1.0 to 1.2 kV / cm , And a DC pulse of 30 를 was added twice to induce cell fusion and activation. Fusion rates were checked 0.5 to 1 hour after the electrical stimulation. At this time, the control group was set as a control group when the same stimulation was given to the oocytes that were not subjected to the micro-manipulation. To the 0.3 M mannitol, 1 mM CaCl ₂ .H ₂ O, 0.1 mM MgCl ₂ .6H ₂ O &lt; / RTI &gt; and 0.5 M HEPES buffer.

<5-2> Cultivation and occurrence of replication column

After the fusion clone prepared in Example <5-1> was selected, 20 to 30 clones were cultured in a 4-well container containing 500 μl of the development medium for 6 days. After completion of the incubation, the cells were stained with 5 쨉 g / ml of Hest 33342, and under the fluorescence microscope, the clone and the unit cell ("the egg is activated by the external stimulus without the modification, ) Were examined. In this case, the case where the microorganisms were cultured by the same method was set as a control group. The culture medium was NCSU-23 (North Carolina State University -23; Petters, RM and Wells, KD., J. Reprod. Fertil. Suppl. , 1993, 48: 61-73) supplemented with 0.4% BSA.

As a result, the fusion rate of 1.0 ㎸ / ㎝ electric stimulation group was 66.1%, that of 1.1 ㎸ / ㎝ treated group was 81.5%, and that of 1.2 ㎸ / ㎝ treated group was 78.5% Fusion rate was significantly lower. However, the fractionation rate was 67.2 to 68.6%, the blastocyst production rate was 14.6 to 16.6%, and the blastocyst cell number was 22.1 to 24.5, showing no difference between treatments with different voltages ( Table 1 )

Effect of electrical stimulation for cell fusion and activation on the development of cloned cells process Voltage (kV / cm) Number of eggs Fusion rate (%) Partitioning rate (%) Incidence of blastocyst (%) Number of blastocyst production (range) Control group 1.0 118 68.4 ± 5.5 17.4 ± 2.5 26.3 ± 2.3 (13-54) 1.1 141 84.0 ± 5.0 12.7 ± 2.3 25.3 ± 2.5 (26-56) 1.2 230 75.2 ± 4.1 19.9 ± 1.9 27.9 ± 1.6 (14-68) Nuclear transfer 1.0 190 66.1 ± 3.1 67.6 ± 5.0 16.6 ± 2.3 22.1 ± 2.3 (17-38) 1.1 224 81.5 ± 3.1 68.6 ± 5.0 15.8 ± 2.3 23.8 ± 2.0 (10-40) 1.2 299 78.5 ± 2.7 67.2 ± 4.3 14.6 ± 2.0 24.5 ± 1.9 (14-41)

In Table 1 ,

,

,

.

From the above results, it can be seen that the method of producing the clone of the present invention has a significantly higher fusion rate and fractionation rate than the conventional method, and thus it is possible to omit the nuclear staining procedure to confirm the presence or absence of fusion.

As described above, since the replication method of the present invention can simplify the cloning process of pigs and produce a highly efficient clone, the method of the present invention can be used for preservation of endangered animals, production of useful protein, And can be usefully used for the production of replication fields for development and supply of deficient organs.

Claims (9)

I) removing the nucleus of the oocyte by inhaling the first polar body and 10-20% of the cytoplasm without staining the nucleus of the oocyte; Ii) injecting a somatic cell into the proliferation of the oocyte in which the first polar body and the nucleus are removed to prepare an oocyte-somatic cell complex; And Iii) cell fusion and activation of said oocyte-somatic cell complex with an electrical stimulation carried out 1 to 3 times at 0.5 to 2 kV / cm, 6 to 150 μs. The method according to claim 1, wherein the inhalation of step i) is carried out using a hydraulic pressure differential using a glass tube having a diameter of 20-40 mu m and a tip structure of 30-60 DEG. The method according to claim 1, wherein the oocyte of step i) is collected from mammals other than humans. 4. The method according to claim 3, wherein the mammal is a species selected from the group consisting of pigs, cows, goats, mice, mice, rabbits, dogs, cats. The method according to claim 1, wherein the somatic cells are collected from mammals other than humans. 6. The method according to claim 5, wherein the mammal is a species selected from the group consisting of pigs, cows, goats, mice, mice, rabbits, dogs, and cats. 7. The method of claim 6, wherein the mammal is a pig. 2. The method according to claim 1, wherein the somatic cells are injected within one minute after the step (i). The method according to claim 1, wherein electrical stimulation is performed twice at 1.0 to 2.0 kV / cm and 30 μs.