CN102766655A - Method for producing somatic cell cloned bovine blastocyst - Google Patents

Abstract

The invention discloses a method for producing somatic cell cloned bovine blastocysts. In this method, bovine ear margin fibroblasts are used as nuclear transplantation donor cells, and mature bovine oocytes cultured in vitro are used as nuclear transplantation recipient cells. The arrangement of oocyte-donor cell-oocyte is adhered and electrically fused to obtain a reconstructed embryo, and the reconstructed embryo is activated and cultured in vitro to obtain a cloned blastocyst. The method can improve the production efficiency of cloned bovine blastocysts.

Description

A method for producing somatic cell cloned bovine blastocysts

technical field

The invention relates to a method for producing somatic cell cloned bovine blastocysts.

Background technique

Mammalian somatic cell cloning refers to the transfer of somatic cell nuclei into enucleated recipient cells (mature oocytes or fertilized eggs), and the new reconstructed embryos are reprogrammed, divided, differentiated and developed according to the donor's genetic information. The more popular somatic cell nuclear transfer methods are manual cloning and micromanipulation.

The efficiency of cloning technology itself is affected by various links, including enucleation of oocytes, injection of somatic cells into oocytes, fusion of enucleated oocytes and somatic cells, activation of reconstructed embryos, and in vitro culture of reconstructed embryos.

Enucleation of mature oocytes in the cloning process is an important link in the nuclear transfer technology, which requires complete enucleation while minimizing damage to the cytoplasm. The enucleation of the manual cloning method uses the cutting method, which causes great damage to the cells and has a high mortality rate; the microinjection method penetrates the operation needle into the cytoplasm of the oocyte, which is more destructive to the cytoskeleton and membrane, and absorbs the oocyte during enucleation. The removed cytoplasm containing chromosomes generally accounts for about a quarter of the total volume of the oocyte, resulting in a large loss of oocyte cytoplasm. In addition, there are fluorescence-guided enucleation method, blind aspiration method and so on. Fluorescence-guided enucleation method, because the use of fluorescent irradiation has certain damage to oocytes, it was reported [Critser et al., 1986] that the irradiation time under fluorescent irradiation should not exceed 20 seconds, otherwise the developmental ability will be reduced or lost. The blind aspiration method refers to the method of McGrath and Solter [McGrath et al., 1983], that is, the nuclear material and part of the cytoplasm in the cell membrane are aspirated together from the position close to the discharge of the first polar body of the oocyte. Because the metaphase plate of mature oocytes in other large animals other than mice and rabbits cannot be seen under the microscope, enucleation is blind. Blind suction enucleation is not entirely successful due to disturbance of granulosa cells or degeneration of the first polar body or aging of the egg, which makes the chromosome position deviate from the first polar body.

Fusion is another important link in the process of animal cloning technology, and the common method is electrical stimulation. The principle of electrofusion is to use a high-voltage electric field to simultaneously punch some micropores on the egg cell membrane and the nucleus donor cell membrane, so that the egg cell membrane and the nucleus donor cell membrane that are close together will fuse through these micropores, and finally introduce the donor nucleus into the enucleation within the egg cytoplasm. The fusion rate in the microinjection method is low, averaging 70-80%, and the failure of fusion leads to the loss of embryos and the reduction of cloning efficiency.

Somatic cell cloning technology has been successful in many breeds of livestock, and thousands of somatic cell cloned cattle have been born around the world today. Nevertheless, the efficiency of bovine somatic cell cloning is still very low. Therefore, it is of great significance to develop a high-efficiency somatic cell cloning bovine blastocyst production method starting from the key links of somatic cell cloning.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for producing somatic cell cloned bovine blastocysts with high efficiency.

The method for producing somatic cell cloned bovine blastocyst provided by the present invention comprises the following steps:

(1) Using bovine ear margin fibroblasts as donor cells for nuclear transplantation;

(2) Bovine oocytes are matured and cultured in vitro, and the oocytes matured in vitro and discharged from the first polar body are used as nuclear transfer recipient cells;

(3) extruding the oocyte obtained in step (2) to enucleate and removing the zona pellucida;

(4) Firstly, an oocyte obtained in step (3) is adhered to a donor cell to form a complex by PHA method, and an alternating current is applied, and another oocyte obtained in step (3) is adhered to the above-mentioned complex. On the body, the arrangement order is oocyte-donor cell-oocyte; finally, it is fused with direct current to construct a reconstructed embryo;

(5) After the reconstituted embryos are activated, they are cultured in vitro to obtain cloned blastocysts.

In the above production method, the generation age of the bovine ear margin fibroblasts is 5-15 generations, and the serum starvation treatment or no serum starvation treatment is performed before the nuclear transplantation.

The bovine oocytes are oocytes collected from bovine follicles with a diameter of 2-8 mm and surrounded by complete and dense cumulus cells.

In the above method, the preparation method of the medium for in vitro maturation culture described in step (2) is to add 10% FBS by volume percentage, 0.01IU/ml of FSH, 0.01IU/ml of LH and 1 μg /ml of E2.

The extruding enucleation in step (3) is to pierce the zona pellucida at the first polar body with a sharp enucleation needle under a microscope, and squeeze out the first polar body and the cytoplasm below it; Bands were removed by pronase digestion.

The interval between the electrofusion in step (4) and the activation of the reconstituted embryo in step (5) is 1-3 hours, preferably 2 hours.

The inventors compared the cleavage rate and blastocyst rate of zona-free cloned bovine embryos whose time intervals between electrofusion and activation were 1 h, 2 h, and 3 h, respectively. The results are shown in Table 1. The formula for calculating the cleavage rate is: cleavage rate=number of cleaved embryos/number of reconstructed embryos after fusion; the formula for calculating the blastocyst rate is: blastocyst rate=number of blastocysts/number of cleaved embryos . The same below.

Table 1. Effect of time interval between electrofusion and reconstituted embryo activation on cleavage rate and blastocyst rate

treatment group Number of eggs processed Cleavage number/rate (%) Number of blastocysts/rate (%) 1h 136 119(88.05±8.06) 37(31.45±3.07) 2h 153 117(78.79±25.67) 53(43.29±13.59) 3h 177 135(76.62±11.67) 40(32.95±8.82)

It can be seen from Table 1 that the cleavage rate of the treatment group with an interval of 2 hours between electrofusion and reconstituted embryo activation was not significantly different from that of the 1h group and the 3h group, but the blastocyst rate was higher than that of the other two groups, suggesting that electrofusion The time interval between the activation of the reconstituted embryo and the reconstituted embryo was 2h can promote the development of the reconstituted embryo without zona pellucida.

In the above method, the activation of the reconstituted embryo in step (5) is the joint activation of A23187 and 6-DMAP.

The in vitro culture medium of the reconstituted embryo is CR1aa, mCR1aa or SOFaaci, preferably mCR1aa culture medium.

The inventors compared the cleavage rate and blastocyst rate of the zona-free cloned bovine embryos in the in vitro culture medium of the above three reconstituted embryos, and the results are shown in Table 2.

Table 2. Effects of different culture media on the development of bovine zona-free nuclear transfer reconstituted embryos

culture medium Number of eggs processed Cleavage number/rate (%) Number of blastocysts/rate (%) CR1aa 102 61/(65.56±26.41) 18/(29.74±1.37) mCR1aa 179 146/(83.17±4.76) 41/(30.62±7.25) SO Faaci 106 42/(37.85±10.44) 11/(26.19±4.12)

It can be seen from Table 2 that there is no significant difference in the blastocyst rate of the reconstituted embryos without zona in the three culture mediums, but the cleavage rate in the mCR1aa culture medium is higher than that of the other two groups, suggesting that there is no clear zone in the mCR1aa culture medium. The developmental rate of cloned bovine embryos is higher.

The present invention combines extrusion enucleation method with zona-free oocyte technology for the first time in the production of somatic cell cloned cattle. Compared with traditional manual cloning and microinjection methods, it not only improves the fusion rate, but also increases the cleavage rate and blastocyst rate. At present, the somatic cell cloned offspring of one Holstein bull has been successfully obtained by using this method, and eight somatic cell cloned cattle will be born in the future.

Description of drawings

Fig. 1 is the extrusion enucleation schematic diagram of oocyte of the present invention;

Fig. 2 is the cytoplasm containing chromosome extruded by extruding and enucleating in the present invention;

Fig. 3 is the fusion process of enucleated and zona-free oocytes and somatic cells of the present invention, from left to right enucleated oocyte-donor cell complex, fused zona-free reconstituted embryo, Fused zona-free reconstructed embryos;

Fig. 4 is the in vitro development process of somatic cell cloned bovine embryos prepared by the present invention; the upper figure is successively 1-cell stage, 2-cell stage, and 4-cell stage from left to right, and the lower figure is successively 8-16 cell stage from left to right , morula stage, blastocyst;

Figure 5 is a schematic diagram of the zona pellucida of 0.5% pronase digested oocytes by manual cloning method;

Fig. 6 is the oocyte cut by the cutting knife in the manual cloning method.

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with specific examples.

In the following examples, the calculation formula of the fusion rate is: fusion rate = number of reconstituted embryos after fusion/logarithm of half eggs before fusion; the calculation formulas of the cleavage rate and blastocyst rate are the same as those described above.

Example 1 Preparation of cloned bovine blastocysts by somatic cell cloning method of the present invention

(1) Preparation of donor cells

Take ear margin fibroblasts of 5-15 generations of Holstein bulls, and when the cells grow to 70-80% confluent, replace with DMEM medium containing 0.5% fetal bovine serum, continue to culture for 3-5 days, and set aside.

(2) Collection and in vitro maturation of oocytes

Collect bovine ovaries from the slaughterhouse, put them in normal saline at about 30°C, and transport them back to the laboratory in time. After the ovary was washed three times with normal saline, the follicles with a diameter of 2-8mm were sucked with a vacuum pump, and the complete and dense cumulus-oocyte complexes (cumulus-oocyte complexes (COCs)) were selected under a stereomicroscope. Wash twice with calcium-magnesium-free PBS solution containing 0.01% PVA, then transfer to TCM199+10%FBS+0.01IU/mL FSH+0.01IU/mLLH+1μg/mL E2 mature culture medium, 50-60 cells/well. Place them in a carbon dioxide incubator at 38.5°C, 5% CO2, and saturated humidity for 16-18 hours. The mature COCs were removed with 0.1% hyaluronidase to remove the granulosa cells, and then the oocytes with complete morphology, uniform cytoplasm and the appearance of the first polar body were selected as nuclear transfer recipients.

(3) Extrusion enucleation of oocytes

Pipette the oocyte with the first polar body into the micromanipulation fluid. Under a 200× microscope, use a sharp enucleating needle to pierce the zona pellucida at the polar body, and squeeze out the first polar body and the cytoplasm below it. The enucleated oocytes were placed into T10 droplets one by one, and the cytoplasmic globules were stained with H33342 to detect whether they contained nuclei and polar bodies according to the one-to-one correspondence. After confirming that the enucleated eggs were washed three times in T10, they were digested with 0.5% pronase to remove the zona pellucida. Then wash it three times with T20 and put it into the incubator for standby. See Figure 1 for the schematic diagram of extrusion and enucleation, and Figure 2 for the extruded cytoplasm containing chromosomes.

(4) Construction of reconstructed embryos - no zona pellucida method

First treat the above-mentioned enucleated, zona-free oocytes in 200 μg/ml phytohemagglutinin (PHA) droplets for 3 to 4 seconds, and then put them into the droplets containing somatic cells for one-to-one adherence As for the cells, those with a medium size and a rounded appearance and a smooth appearance were selected as donor cells. Apply 3V alternating current (AC), so that the arrangement direction of the somatic cell-oocytoplasm complex in the fusion tank is automatically perpendicular to the electrode, and then another enucleated oocyte without zona pellucida is also adhered to it under the action of the alternating electric field. On the complex, the arrangement order is oocyte-donor cell-oocyte, and the arrangement direction is perpendicular to the electrodes. Finally, 50V/mm, 9μs, 1 pulse of direct current (DC) was applied for electrical fusion, and the fusion rate was counted. The fusion process is shown in Figure 3.

(5) Activation and in vitro culture of reconstituted embryos

After 2 hours of electrofusion, the reconstituted embryos were transferred to the mCR1aa culture medium containing an appropriate amount of A23187 for 5 minutes, washed three times, and then transferred to the mCR1aa culture medium containing 2mM 6-DMAP for further activation and culture for 6 hours, and finally transferred to the mCR1aa culture medium for in vitro nourish. The reconstituted embryos without zona pellucida were cultured in vitro in microcavities, and the microcavities were made according to the method reported by Vajta. The reconstituted embryos were cultured in vitro for about 48 hours, the medium was changed and the cleavage rate was checked; the development of the reconstituted embryos was checked on 7-8 days, and the blastocyst rate was counted.

The in vitro development process of the somatic cell cloned bovine embryo prepared by the above method is shown in FIG. 4 .

Example 2 Preparation of cloned blastocysts by manual cloning

Among them, the preparation of donor cells, the collection of oocytes and in vitro maturation culture, the activation of reconstituted embryos and the in vitro culture steps are the same as the corresponding steps in Example 1, oocyte enucleation and zona-free oocyte and body The fusion process of cells is as follows:

To core manually:

Use 0.1% hyaluronidase to remove the granulosa cells of the oocyte-cumulus cell complexes that have been matured in vitro for about 18 hours, and then place them in the maturation solution added with 5% decarboxycolchicine (Demecolcine, DC) for 2 hours Then select the oocytes with homogeneous cytoplasm that are expelled from the polar body and digest the zona pellucida with 0.5% pronase protease. Reserve in droplets. See Figures 5 and 6.

Fusion of zona-free oocytes with somatic cells:

Firstly treat the half egg in 200μg/ml phytohemagglutinin (PHA) droplet for 3-4s, then put it into the droplet containing somatic cells for one-to-one adherent somatic cells, select medium-sized volume, recover Round smooth ones were used as donor cells. Apply 3V alternating current (AC), so that the arrangement direction of the somatic cell-oocytoplasm complex in the fusion tank is automatically perpendicular to the electrode, and then another oocytoplasm is also adhered to the complex under the action of the alternating electric field to make it align The sequence is egg-somatic cell-egg, and the arrangement direction is guaranteed to be perpendicular to the electrodes. Finally, 50V/mm, 9μs, 1 pulse of direct current (DC) was applied for electrofusion. The fusion was detected 30 min after the fusion, and the fusion rate was counted.

The successfully fused reconstituted embryos were activated and cultured in vitro, the steps of which were the same as those in Example 1, during which the cleavage rate and blastocyst rate were counted.

Example 3 Preparation of cloned blastocysts by microinjection

Among them, the preparation of donor cells, collection of oocytes and in vitro maturation culture, activation of reconstituted embryos and in vitro culture steps are the same as the corresponding steps in Example 1. The process of enucleation of oocytes and construction of reconstituted embryos is as follows:

Microenucleation:

Transfer the oocyte with the first polar body into the operating solution H199+7.5ug/mL cytochalasin B droplet, and use a 20 μm diameter glass pipette (with a tip) to remove the first polar body and The chromosomes in the oocyte below it are sucked out together. The operated oocytes were transferred to the corresponding drop in another dish, and the cytoplasm containing chromosomes was stained with Hoechst33342 for 5 minutes, and the enucleation rate was checked under UV light, and the enucleated oocytes were washed three times in T20 solution Finally, put it in a 38.5°C, 5% CO2 box for later use.

Construction of the reconstructed embryo:

(1) Micronucleation

Transfer the enucleated oocytes and appropriate amount of donor cells into the operating drop, and use a hanging tip with an inner diameter of 20-25 μm to absorb a round, smooth donor with a diameter of 15-20 μm and strong refraction The cells are injected into the perivitelline space of the enucleated oocyte through the incision left during enucleation on the zona pellucida, and the injected donor cells are as close as possible to the egg cell membrane, and the reconstructed embryos are placed in the embryo culture medium to recover for 1 to 2 hours. .

(2) Electrofusion of reconstructed embryos

Put the recovered reconstituted embryos into the fusion solution in batches to balance for 2 minutes, and rotate the oocytes in the fusion tank with an egg-pushing needle, so that the contact surface between the donor cells and the oocytes is perpendicular to the direction of the electric field, and at the same time apply a field strength 2.2kV·cm ^-1 , the pulse time is 20μs, and the number of pulses is 1 DC pulse for fusion (ECM-2001BTX). After 30 minutes of recovery, the fusion rate is counted.

The fused reconstituted embryos were selected for further activation treatment and cultured in vitro, during which the cleavage rate and blastocyst rate were counted.

The fusion rate, cleavage rate and blastocyst rate of above-mentioned embodiment 1,2,3 clone blastocyst preparation method are as shown in table 3:

Table 3. Effects of different nuclear transfer methods on transgene cloning efficiency

nuclear transfer method Reconstruct embryo number fusion rate Cleavage rate Blastocyst rate The method of the present invention 324 95.24%±1.58 90.56%±2.74 38.35%±4.13 manual cloning 153 90.33%±2.29 82.97%±2.49 32.36%±1.39 Microinjection 251 72.94%±7.92 89.10%±5.76 33.59%±2.25

As can be seen from Table 3, the fusion rate, cleavage rate, and blastocyst rate of the method of the present invention are higher than those of the manual cloning method and the microinjection method, indicating that the method of the present invention has a higher bovine somatic cell cloning efficiency.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (9)

1. method of producing somatic cell clone ox blastaea may further comprise the steps:

(1) be the nuclear transplantation donorcells with the ears of an ox or cow edge inoblast;

(2) bovine oocyte is carried out maturation in vitro and cultivate, vitro culture is the ripe and ovocyte of discharging first polar body is as the nuclear transplantation recipient cell;

(3) step (2) gained ovocyte is pushed stoning and removes zona pellucida;

(4) earlier one piece of step (3) gained ovocyte and one piece of donorcells are sticked the formation complex body with the PHA method; Impose alternating-current; Again another piece step (3) gained ovocyte is attached on the above-mentioned complex body, it is put in order be ovocyte-donorcells-ovocyte; Impose direct current at last it is merged, make up the reconstruct embryo;

(5) after said reconstruct embryo activates, carry out vitro culture DCRP blastaea.

2. the method for claim 1 is characterized in that, said the ears of an ox or cow edge fibroblastic age in generation is 5-15 generation, before nuclear transplantation, handles through serum starvation or handles without serum starvation.

3. the method for claim 1 is characterized in that, said bovine oocyte be by diameter be gather in the ox ovarian follicle of 2-8mm, by the ovocyte of the cumulus cell parcel of complete densification.

4. the method for claim 1; It is characterized in that the preparation method of the substratum that the said maturation in vitro of step (2) is cultivated is that the interpolation volume percent is 10% FBS in the TCM199 substratum; 0.01IU/ml FSH, the E2 of the LH of 0.01IU/ml and 1 μ g/ml.

5. the method for claim 1 is characterized in that, the said extruding stoning of step (3) is to needle the zona pellucida at first polar body place at microscopically with top-notch kernel removing needle, and the kytoplasm of first polar body and below thereof is squeezed remove; The said zona pellucida that goes is to remove with pronase digestion.

6. the method for claim 1 is characterized in that, said electricity merge with said reconstruct embryo activated pitch time be 1-3h.

7. method according to claim 6 is characterized in that, said electricity merge with said reconstruct embryo activated pitch time be 2h.

8. method according to claim 1 is characterized in that, said reconstruct embryo's activation is the activation of uniting of A23187 and 6-DMAP.

9. the method for claim 1 is characterized in that, said reconstruct embryo's vitro culture liquid is CR1aa, mCR1aa or SOFaaci.

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