KR20050034187A - Method of efficient surgical transfer of porcine nuclear transfer embryos - Google Patents

Abstract

The present invention relates to a method for transplanting pig cloned eggs into surrogate sows, and more particularly, 1) culturing the cloned eggs in a fertilized egg transport kit; 2) anesthetizing surrogate with pentotalsodium and isofran; And 3) transplanting porcine cloned eggs into surrogate sows comprising the step of transplanting the cloned eggs of step 1 to surrogate sows having an ovary immediately before ovulation or within 2 days after ovulation. The method of transplanting pig cloned eggs of the present invention can improve the production efficiency of cloned pigs by improving the pregnancy problems after transplantation by improving the conventional problems in transplantation of cloned eggs, and also transplanting not only cloned eggs but also general pig fertilized eggs. It can also be applied to increase the production efficiency of pigs.

Description

Method of efficient surgical transfer of porcine nuclear transfer embryos

The present invention relates to a method of transplanting pig cloned eggs into surrogate sows, and more particularly, to improve the conventional problems in transplanting cloned eggs, thereby increasing the production efficiency of cloned pigs by increasing pregnancy efficiency after transplantation. A pig cloned egg relates to a method of transplanting the surrogate sows.

Cloning refers to the creation of genetically identical organisms. Nuclear transfer is a technique that has attracted new attention due to the recent rapid development of molecular biology as a method of cloning in eukaryotes. Nuclear transfer of early, but using the blastomeres of embryos as a source of nuclei (Prather, et al, B iol Reprod, 1987, 37:...... 856-866; Prather, et al, Biol Reprod, 1989, 41 : 414-418) Recently, somatic nuclei have been used.

Somatic cloning is a technique in which differentiated somatic cells are inserted into a nucleus from which the nucleus has been removed and activated, and then generated in the same manner as a normal fertilized egg. In general, inhalation of the cytoplasm removes the nucleus of an egg and implants somatic cells into the nucleus from which the nucleus has been removed. And then applying electrical stimulation to fuse and activate the oocyte-somatic complex. Such cloning technology can be widely used in research in basic sciences including developmental biology, and is expected to contribute greatly to various medical and pharmaceutical fields such as production of useful proteins, development of disease models, and transplantation of organs. It can be said that the availability is very large.

The fertilized egg transplantation of pigs was developed to improve the breeding efficiency of pigs by supplying large quantities of excellent breed pigs in a short period of time. Recently, as pigs emerged as target animals for the production of expensive therapeutic proteins and replacement organs through cloning and transformation, There is a growing interest in the transplantation of porcine cloned eggs.

Since the birth of the first somatic cloning animal, Dollmut (I. et al., Nature , 1997, 385: 810-813), the field of somatic cloning has evolved a great deal (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), goats (Bagusi, A. et al., Nat. Biotechnol ., 1999, 17: 456-461) have been successfully cloned. However, compared to other species, pigs have been relatively poorly studied in fertilized eggs, and due to various difficulties, such as physiological characteristics in which at least four fetuses are implanted in the uterus to maintain pregnancy, the pigs have been successfully replicated relatively late among major livestock. (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). Subsequently, the first successful transgenic cloned pig was born by injecting an external gene into pig somatic cells (Park, KW. Et al., Anim. Biotechnol ., 2001, 12: 173-181). Cloned pigs knocked out in somatic cells were also successful (Lai, L. et al., Nat. Biotechnol. , 2002 Mar, 20 (3): 251-255). However, despite the rapid development of the porcine somatic cloning field, the efficiency of cloning is still low at 1-5%, and also various health and physical symptoms after childbirth, such as mortality of the fetus in pregnancy and maternal hypertrophy of the fetus. Abnormalities have been reported (Cibelli JB. Et al., Science , 1998, 280: 1256-1258; Hill JR et al., Theriogenology , 1999, 51: 1451-1465; Carter DB et al., Cloning Stem Cells , 2002, 4: 131-145; Park KW et al., Biol. Reprod. , 2002, 66: 1001-1005) Many studies are needed to improve the efficiency of replication.

On the other hand, studies on the timely transplantation of cloned eggs in somatic cell cloning of pigs have not been carried out. Currently, most researchers are transplanting to surrogate sows within 1-2 days after the production of cloned pigs, but the study of various conditions suitable for transplantation is insignificant.

In order to solve the above problems in the production of cloned pigs, the present inventors have examined the transport of cloned eggs, selection of cloned egg transplanters, and surgical operations in the production of cloned pigs. Efforts have been made to improve the results of the present invention by establishing optimal pig cloning transplant conditions.

It is an object of the present invention to provide an optimal transplantation method for transplanting pig cloned eggs into surrogate sows.

In order to achieve the above object, the present invention

1) culturing the cloned eggs in a fertilized egg transport kit;

2) anesthetizing surrogate with pentotalsodium and isofran; And

3) Provided is a method for transplanting pig cloned eggs into surrogate sows comprising the step of transplanting the cloned eggs of step 1 to surrogate sows having an ovary immediately before ovulation or within 2 days after ovulation.

Hereinafter, the present invention will be described in detail.

In order to achieve the above object, the present invention

1) culturing the cloned eggs in a fertilized egg transport kit;

2) anesthetizing surrogate with pentotalsodium and isofran; And

3) Provided is a method for transplanting pig cloned eggs into surrogate sows comprising the step of transplanting the cloned eggs of step 1 to surrogate sows having an ovary immediately before ovulation or within 2 days after ovulation.

In the above, the incubation time of the cloned eggs of step 1 is preferably within 5 hours.

In addition, the dose of pentotalsodium in step 2 is preferably 1.0 to 2.0 g, more preferably 1.5 g, and isopran preferably inhales 2 to 5% of isopran with 5% oxygen. .

In stage 3, the ovary was divided into three stages according to the ovulation time and time of ovulation, the follicle was enlarged immediately before ovulation, or the ovulation was large and the reddish ovary (OD0 stage) and ovulation had passed. It is selected from the group consisting of ovary (OD1 stage) with bleeding point and ulceration point, ovary with ovulation point and ovary with a little development of luteal mass, and papillary ovary (OD2 stage). desirable.

In the present invention, in the production of cloned pigs, the process of remote transfer of cloned eggs, timely review of cloned egg transplantation time, and surgical operations were improved to enhance the transfer efficiency of cloned eggs and the production efficiency of cloned pigs.

Since the cloned eggs are relatively weak, it is preferable to remove them from the incubator in a short time when transplanting them into surrogate sows. This is not a problem when the transplant is near the laboratory, but when it is necessary to move to the farm due to various circumstances, it is highly likely to harm the fertilized eggs in the process of transporting the cloned eggs to the transplant site. It can be hard to implant in your money, and even after implantation, the fetus may not develop properly. In the present invention, in order to solve the above problems, an embryo transport kit (embryo transport kit, Mititub, Germany) was used. The fertilized egg transport kit of the present invention is a device for maintaining a temperature for a predetermined period of time by specifying a temperature of 35 ℃ to 42 ℃, consisting of a cylindrical reservoir and a charger for maintaining the temperature of the reservoir. In the present invention, the cloned eggs were incubated in the kit for 5 hours and again cultured in a general incubator, and in vitro development of the cloned eggs did not show a significant difference from the control group (see Table 1). The results suggest that transporting cloned eggs within 5 hours using the fertilized egg transport kit does not interfere with in vitro development of the cloned eggs.

Pig embryo transplantation has been developed a lot, and recently, it has been possible to produce cloned pigs by transplanting cloned fertilized eggs. However, it is not clear at what time of day the transplant is time for surrogate sow, and most researchers now transplant to surrogate sows on the day or within the day of cloning. In the selection of surrogate sows, transplantation is based on estrus, but since the duration of estrus ranges from 12 to 96 hours, it may be more appropriate to determine the ovarian ovulation status rather than estrus. Therefore, in the present invention, the experiment was performed after dividing the ovary into three stages of OD0, OD1 and OD2 according to the state of ovulation point (see FIG. 1). In the above, OD0 is before and after ovulation, OD1 is when the ovulation point is any after ovulation, and OD2 is when the ovulation point is completely healed, and it indicates the time of bloat. As a result, the initial pregnancy (30 days of pregnancy) was confirmed, 2 in OD0 and 1 in OD1 and OD2 respectively, and OD1 regressed 64 days in pregnancy and the other three had cesarean section at 114 days of pregnancy. (See Table 2). This fact indicates that surrogate sows are suitable for transplantation on day 1 of estrus when transplanting 1-2 days old cloned eggs, and implantation before and after ovulation may increase pregnancy rates based on ovarian status.

In the present invention, pentotal sodium and isopran were used to anesthetize surrogate sows. Pentotalsodium is one of the thiobarbituric acid compounds synthesized by Volwiler and Jabern in 1935. It is an ultrashort type of anesthetic and it relieves sedation, sleep and anesthesia by reversibly inhibiting the brain stem rehabilitation system of the central nervous system. Action. When pentotalsodium is administered to the ear vein, the surrogate sows lose their mind in one minute and fall down, and after a few minutes they start to wake up slowly, so you should inhale 2-5% isofran with 5% oxygen in the shortest possible time. Using the anesthesia method as described above can easily anesthetize the pig, and the pig does not break during the operation can be less pain to the pig. After the operation is over and the supply of isopran is cut off, the pig can recover from consciousness in minutes and walk within 30-60 minutes. This fact can be said that the method of anesthesia is less stress on the pig. This anesthesia method is generally applicable in the operation of pigs, especially if the purpose of obtaining live in pigs after surgery, such as cloning egg transplantation will be desperately needed.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Cloned Egg Using Porcine Somatic Cells

<1-1> Isolation of Porcine Somatic Cells

After gestation of a 35-day-old pig fetus with a razor blade, the cells were incubated for 5 minutes in DMEM medium (bio-whittaka. Inc) to which trypsin-EDTA (Gibco. Inc) was added. The supernatant was centrifuged (1,000 rpm, 5 min), incubated in DMEM medium containing 10% Fecal Calum Serum (Hyclone. Inc), and passaged after trypsin treatment when the cells reached 90% confluency. Some of them were used for nuclear transfer and others were cryopreserved.

<1-2> Oocyte Collection and Culture

Ovaries of unpolished pigs were collected from slaughterhouses and stored in 35-39 ° C. and 0.9% physiological saline. An egg was collected by attaching an 18 gauge needle to a 10 ml syringe and sucking the follicular fluid from the follicle of 2 to 6 mm in diameter. The eggs were washed in mature culture medium, and 50 to 60 eggs were added to 500 µl culture and incubated for 42 to 46 hours.

At this time, the mature culture medium in TCM 199 (31100035; Gibco, Grand Island, NY) 0.1% polyvinyl alcohol (polyvinyalcohol), 3.05 mM glucose (D-glucose), 0.91 mM sodium pyruvate, 0.57 mM cysteine (cysteine), 0.5 μg / ml LH (Sigma Chemical Co. St. Louis, MO), 0.5 μg / ml FSH (Sigma), 10 ng / ml epidermal growth factor (Sigma), 75 μg / ml penicillin Prepared by adding G and 50 μg / ml streptomycin.

<1-3> micromanipulation

After culturing the oocytes harvested in Example <1-2> for 5 to 10 minutes in a microalgae culture solution, the porcine somatic cells isolated in Example <1-1> were added to the culture solution. At this time, the microalgae culture was prepared by adding 0.3% BSA and 7.5 μg / ml CB (cytochalasin B) to TCM 199.

In order to remove the nucleus of an egg, a 30 micrometer diameter microglass tube whose tip was sharply ground to 30-60 degrees was prepared. First, the pole body of the egg was fixed at 1 o'clock, and the microglass tube was inserted into the pole body. Hydraulic pressure was used to inhale about 10-20% of the pole body and the cytoplasm around the pole. Since most of the nuclei are located around the pole, most of the nuclei of the eggs were removed. Thereafter, the porcine somatic cells prepared in Example 2 were sucked using the microglass tube, and the microglass tube was placed in the gastric cavity of the egg from which the nucleus was removed, and the somatic cells were injected into the gastric cavity of the egg using hydraulic pressure.

<1-4> Cell fusion and activation

After the micromanipulation of Example <1-3>, the nuclear transfer embryos were placed in the activated culture medium, and the egg-somatic cell complexes were placed between the platinum electric wires spaced 1 mm apart, and then subjected to 1.1 ㎸ with a fusion machine (BTX Electro-Cell Manipulation 2001). DC pulse of 30 cm / cm / cm was added twice to induce cell fusion and activation. The fusion rate was checked 0.5 to 1 hour after the electrical stimulation was applied. The activated culture was prepared by adding 1 mM CaCl ₂ .H ₂ O, 0.1 mM MgCl ₂ .6H ₂ O, and 0.5M HEPES buffer to 0.3 M mannitol.

<1-5> Cultivation and development of cloned eggs

After selecting the fused cloned eggs prepared in Example <1-4>, 20-30 cloned eggs were incubated for 6 days in a 4-well container containing 500 μl of the developing culture solution. After completion of the culture, 5 μg / ml of hex 33342 was stained and the number of nuclei was examined under a fluorescence microscope. The development culture was prepared by adding 0.4% BSA to NCSU-23 (Northern Carolina State University-23; Petters, RM. And Wells, KD., J. Reprod. Fertil. Suppl. , 1993, 48: 61-73) medium. It became.

Example 2 Establishment of Optimal Cloning Embryos Transplantation Conditions

<2-1> Transport of cloned eggs

The effects of storage of cloned eggs in fertilized egg transport kit on in vitro development of cloned eggs were examined. To this end, the present inventors transferred the cloned eggs prepared in Example 1 to the tube containing the embryo culture on the morning of transplanting, and then put them in a fertilized egg transport kit (embryo transport kit, Minitube) to transport to the transplant facility. At this time, the temperature in the kit was maintained at 38.5 ?? and the D0 stored in the transport kit on the day of making the cloned eggs, the cloned eggs were made and stored in the transport kit for 1 day in the transport kit on the D1, 2nd day. The stored thing was called D2. The cloned eggs were stored in the kit for 5 hours and then cultured again from the incubator to the blastocyst. As a control, the cloned eggs were prepared and not cultured in the fertilized egg transport kit.

<2-2> Surgical Transplantation of Cloned Eggs

Every morning, the vulva of the pig was observed to be red, swollen, and burned from the back. It was estimated that the estrus had come to heat, and when the pig was in contact with it, it was considered that the estrus had not avoided. The embryos were transplanted the day after estrus.

The upper jaw of the pig was fixed with a string, and a rubber band was filled in one ear to block the flow of blood, and then a needle was inserted into the vein. When blood began to flow through the needle, a syringe was connected to the needle, and 1.5 g of Pantotalsodium (Supplied Pharmaceuticals) was dissolved in 30 ml of distilled water for injection. The pigs were placed on the operating table, connected with an inhalation anesthesia in the nose, and then inhaled 2-5% isopran. The pig's lower abdomen hair was removed with a shearing machine and sterilized with betadine and alcohol. 100-200 cloned eggs transported in Example <2-1> were prepared in NCSU23 culture solution containing 0.4% BSA, and 1 mL syringes were connected to a catheter (Tom Cat Catheter, Sherwood, USA) to inhale the cloned eggs. The catheter was then inserted into the fallopian tube and cloned eggs were injected. After the operation, the surrogate sows were placed in the pig room and checked until recovery. On day 35 after the cloned embryos were transplanted into surrogate sows, pregnancy was examined using an ultrasound diagnostic device (Mysono 201, Medison Co. Korea).

As a result, the splitting rate and blastocyst incidence rate of the treatment group were 60.1-64.6% and 7.3-13.6%, respectively, which showed no significant difference from 64.6% and 12.3% of the control group. The number of blastocysts was also 18.4-22.5, showing no difference from 21.2 of the control group (Table 1).

Effect of Replica Egg Transport on In Vitro Development of Replica Eggs Number of embryos % Split Blastocyst incidence (%) Blastocyst Nucleus (Range) Control 247 64.6 ± 3.0 12.3 ± 2.3 21.2 ± 1.0 (14-44) D0 111 63.4 ± 3.5  7.3 ± 2.8 20.8 ± 2.0 (18-26) D1 154 64.1 ± 3.0 12.8 ± 2.3 18.4 ± 1.3 (15-22) D2 190 60.1 ± 2.8 13.6 ± 2.2 22.5 ± 1.2 (14-44)

In Table 1 above,

Indicates.

In addition, the inventors identified the ovulation point in the ovary and classified into three types according to the time elapsed after ovulation and the shape of the ovary. Immediately before ovulation, the follicle was enlarged or ovulated, and the ovulation site was large and reddish. Ovulation progressed, the ovulation site was slightly healed, but red blood redness and ovulation was almost hyperused to OD1, the ovulation point was closed and the corpus luteum developed a little, and the papillary shape was OD2 (Fig. 1). As a result, cloned eggs were transplanted into 14 surrogate sows and 4 pregnant women were obtained. Depending on the ovary, two OD0 animals were found, one OD1 and one OD2 (Table 2).

Transplantation result of cloned egg Surrogate sow number OD Elapsed days Number of embryos Pregnancy (30 days) delivery 35-36 One 2 140 33-185 One 2 152 31-157 2 2 161 Did 3 heads 31-81 2 2 100 31-161 2 2 111 20-25 2 2 102 19-194 One One 190 19-55 0 2 134 Did 5 heads 31-159 2 2 193 18-164 One One 124 Did 64 days pregnant 36-51 2 1, 2 156 (90 + 66) 32-135 One 1, 2 157 (91 + 66) 34-135 0 1, 2 176 (94 + 82) Did 4 heads 32-17 2 1, 2 174 (93 + 81)

Days elapsed: Number of days of in vitro culture from cloned eggs to transplantation

As described above, the method of transplanting pig cloned eggs of the present invention can improve the production efficiency of cloned pigs by improving the pregnancy efficiency after transplantation by improving the conventional problems in transplantation of cloned eggs, as well as cloned eggs. In addition, it can be applied to the transplantation of common pig fertilized eggs can increase the production efficiency of pigs.

Figure 1 is a picture showing the classification of ovary according to the ovulation after ovulation, the ovary (A, OD0) in the stage of ovulation is large and red erythema due to ovarian hypertrophy or ovulation, just before ovulation, ovulation point The site is slightly healed, but it is still red, and the ovulation point is almost hypertrophic ovary (B, OD1) and the ovulation point is closed, and the corpus luteum develops a little and is enlarged, and the papilla-like ovary (C, OD2) is shown.

Claims (5)

1) culturing the cloned eggs in a fertilized egg transport kit; 2) anesthetizing surrogate with pentotalsodium and isofran; And 3) Transplanting pig cloned eggs into surrogate sows comprising the step of transplanting the cloned eggs of step 1 to surrogate sows having an ovary immediately before ovulation or within 2 days after ovulation. The method according to claim 1, wherein the incubation time of the cloned eggs of step 1 is within 5 hours. The method of claim 1, wherein the dose of pentotalsodium in step 2 is 1.0 to 2.0 g / horse. The method of claim 1, wherein the isopran of step 2 is inhaled with 2% to 5% of isopran with 5% oxygen. According to claim 1, the ovary of step 3 is just before ovulation, the follicle is enlarged or ovulation is large, the ovulation area is large, red ovary, ovulation elapsed a little bit of ovulation point, but still bleeding red and ovulation point is almost A method characterized in that it is selected from the group consisting of ovary and ovulation point of the ulcer state, the development of the corpus luteum, the enlargement of the luteum, and the papillary ovary.