CN106591374B - A method for improving the developmental efficiency of porcine somatic cell nuclear transfer embryos - Google Patents

Abstract

The invention discloses a method for improving the developmental efficiency of pig somatic cell nuclear transfer embryos. The present invention provides the application of chaetocin, which is at least one of the following (a1)-(a5): (a1) preparing nuclei donor cell culture fluid for animal somatic cell nuclear transplantation; (a2) preparing for animal body Reconstituted embryo culture medium for nuclear transfer; (a3) cultivating nuclear donor cells for animal somatic cell nuclear transfer; (a4) cultivating reconstituted embryos for animal somatic cell nuclear transfer; (a5) promoting reconstitution in animal somatic cell nuclear transfer Embryo formation blastocyst. The invention promotes the reprogramming of somatic cell nuclear transfer by using the small molecule drug chaetocin to treat the donor cells or reconstructed embryos in somatic cell nuclear transfer, and significantly improves the blastocyst development rate and blastocyst cell number of cloned embryos. Ultimately improve the overall efficiency of porcine somatic cell nuclear transfer technology. The invention has great significance for improving the overall efficiency of the pig somatic cell nuclear transplantation technique.

Description

A method for improving the developmental efficiency of porcine somatic cell nuclear transfer embryos

technical field

The invention relates to the fields of embryo development and animal biotechnology, in particular to a method for improving the development efficiency of pig somatic cell nuclear transfer embryos.

Background technique

The technology of somatic cell nuclear transfer was born in 1997, and it was first successfully used in sheep. The main method is to transfer fully differentiated nucleated donor cells into enucleated oocytes to form reconstructed embryos through micromanipulation and cell fusion technology, and then activate the reconstructed embryos for culture and transplantation, and finally obtain a complete individual. Because the obtained individual has the same genotype as the donor cell, this technique is also called somatic cell cloning technique. Due to its unique technical advantages, somatic cell cloning technology has great application value in animal breeding, disease model construction, endangered animal protection, therapeutic cloning, and pharmaceutical protein production. Since the birth of the first cloned sheep "Dolly" in 1997, somatic cell cloning technology has been successfully used in pigs, cows, dogs, rabbits, cats and other mammals. Although after two decades of development, the cloning technology has been relatively mature, and there is a set of overall technologies including oocyte collection, culture, donor cell isolation, culture, egg activation, embryo in vitro culture and embryo transfer, but so far, The overall efficiency of pig somatic cell cloning is still very low, about 1%. In addition, cloned pigs often have unsound developmental abnormalities, such as large tongues and hypoplastic lungs.

Contents of the invention

The purpose of the present invention is to provide a method for improving the developmental efficiency of pig somatic cell nuclear transfer embryos.

The present invention provides the application of chaetocin, which is at least one of the following (a1)-(a5):

(a1) preparing a nucleus-donor cell culture medium for animal somatic cell nuclear transplantation;

(a2) preparing a reconstituted embryo culture medium for animal somatic cell nuclear transfer;

(a3) cultivating nuclear donor cells for animal somatic cell nuclear transfer;

(a4) culturing reconstituted embryos for animal somatic cell nuclear transfer;

(a5) Promote the formation of blastocysts from reconstituted embryos in animal somatic cell nuclear transfer.

The invention also protects a nucleus donor cell culture solution for animal somatic cell nucleus transplantation, which contains 10nM chaetocin.

The nucleus-donating cell culture fluid includes chaetin and basal cell culture fluid.

The basic cell culture medium can specifically be DMEM culture medium.

The nuclei-donor cell culture solution also includes 15% (volume percentage) fetal bovine serum.

The nucleus-donor cell culture solution also includes 100 U/mL ampicillin and 100 U/mL streptomycin sulfate.

The invention also protects a reconstituted embryo culture solution for animal somatic cell nuclear transfer, which contains 0.5nM chaetocin.

The reconstituted embryo culture fluid includes chaetocin and basic embryo culture fluid.

The basic embryo culture medium can specifically be PZM-3 culture medium.

The present invention also protects a method for nuclear transplantation of animal somatic cells, comprising the following steps: cultivating the nuclei donor cells with any of the nuclei donor cell culture fluid described above.

The culture time may specifically be 24 hours.

The method also includes taking the nucleated donor cells that have been cultured for preparing reconstituted embryos.

The preparation method of the reconstituted embryo can specifically be: injecting a nucleated donor cell cultured by the above method into the perivitelline space of the enucleated oocyte, electric shock fusion and activation of the reconstituted embryo.

The present invention also protects a method for nuclear transfer of animal somatic cells, comprising the following steps: cultivating reconstituted embryos with any one of the reconstituted embryo culture fluids described above.

The method includes two stages of culture: in the first stage, the reconstituted embryos are cultivated with any one of the above-mentioned reconstituted embryo culture fluids; in the second stage, the reconstituted embryos are cultivated in the culture fluid that does not contain chaetin.

The chaetocin-free culture solution can specifically be any one of the basic embryo culture solutions described above.

The first stage is 14-16h, specifically 16h.

The preparation method of the reconstituted embryo can specifically be: injecting a nucleus donor cell into the perivitelline space of the enucleated oocyte, electric shock fusion and activation of the reconstituted embryo.

The conditions for any one of the electric shock fusion mentioned above can specifically be: a direct current pulse of 150v/mm, 100μs, 2DC.

The preparation method of any one of the "enucleated oocytes" mentioned above can specifically be: removing the first polar body and nucleus of the animal mature oocyte by blind aspiration.

Any of the above-mentioned donor cells can specifically be animal fetal fibroblasts.

The method for preparing animal fetal fibroblasts may specifically include the following steps (b1) and (b2):

(b1) Take the isolated fetus, cut off the head, limbs and viscera, cut up the remaining part and transfer it to a petri dish, and cultivate it in an incubator at 37°C, saturated humidity, and 5% _CO for 4-6 hours;

(b2) After completing step (b1), add DMEM culture fluid containing 15% (volume percentage) fetal calf serum to the culture dish for cultivation and passage, instead of passage to the 3rd-5th generation (specifically, passage 3) of contact-inhibited fetal fibroblasts.

The preparation method of any one of the animal mature oocytes described above may specifically include the following steps: (c1)-(c3):

(c1) Take the isolated animal ovary, extract the follicular fluid in the follicle, and select the cumulus oocyte complex (COCs) wrapped with 3-5 layers of cumulus cells;

(c2) Transfer the cumulus-oocyte complexes (COCs) obtained in step (c1) to IVM solution, and culture them in an incubator at 39° C., saturated humidity, and 5% CO ₂ for 42 hours.

(c3) After step (c2) is completed, cumulus cells are removed after digestion with 0.1% hyaluronidase, and mature oocytes with the first polar body and uniform and dense cytoplasm are selected.

Described IVM liquid is made up of solvent and solute, and the concentration in described solute and IVM liquid is: described solute and its concentration in IVM liquid are: 199 culture medium 0.987g/100mL, follicular fluid 10mL/100mL, epidermal growth Factor 1 μg/100mL, follicle-stimulating hormone 50mg/100mL, luteinizing hormone 50mg/100mL, cysteine 7μg/100mL; the solvent is pure water.

Any of the animals mentioned above can specifically be a mammal, and more specifically can be a pig.

Chaetocin is a natural metabolite isolated from Chaetomium fungi, which has a certain inhibitory effect on the growth of tumor cells, so it is used as a potential tumor treatment drug. Chaetocin has not been used in the field of somatic cell reprogramming, and there is no report on the use of chaetin to promote embryonic development.

The present invention uses the small-molecule drug chaetocin to treat the nucleus-donor cells or reconstructed embryos in somatic cell nuclear transfer, thereby promoting the occurrence of somatic cell nuclear transfer reprogramming, and can significantly improve the blastocyst development rate and blastocyst development rate of cloned embryos. Cell number, ultimately improving the overall efficiency of porcine somatic cell nuclear transfer technology. The invention has great significance for improving the overall efficiency of the pig somatic cell nuclear transplantation technique.

Description of drawings

Figure 1 shows the effect of chaetocin on the activity of pig fetal fibroblasts.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

Medium 199: Gibco, Cat. No.: 31100-035.

Epidermal Growth Factor: Promega, Cat. No.: G5021.

Follicle-stimulating hormone: Sigma, Cat. No.: F8174.

Luteinizing hormone: Sigma, Cat. No.: L5269.

IVM liquid (in vitro maturation culture medium) is made up of solvent and solute, and described solute and its concentration in IVM liquid are: 199 culture medium 0.987g/100mL, follicular fluid 10mL/100mL, epidermal growth factor 1μg/100mL, follicle-stimulating Hormone 50mg/100mL, luteinizing hormone 50mg/100mL, cysteine 7μg/100mL; the solvent is pure water.

Chaetin: Sigma, product number: C9492; the molecular formula of chaetin is C ₃₀ H ₂₈ N ₆ O ₆ S ₄ , CAS number: 28097-03-2, and the structural formula is as follows:

DPBS buffer: Sigma, catalog number: D5652.

Embodiment 1, the in vitro maturation culture of porcine oocyte

Take the pig ovary from the slaughterhouse, wash it three times with normal saline containing 100U/mL penicillin and 100U/mL streptomycin, and then use a 10mL disposable syringe and a 18-gauge needle to extract follicles with a diameter of 3-6mm Cumulus-oocyte complexes (COCs) wrapped with 3-5 layers of cumulus cells were selected from the liquid. COCs were rinsed twice with saline containing 100U/mL penicillin and 100U/mL streptomycin, then transferred to IVM solution, and cultured at 39°C, saturated humidity, and 5% _CO2 incubator for 42h.

Embodiment 2, the separation and cultivation of pig fetal fibroblast

Pig fetal fibroblasts were prepared according to the following steps in turn:

1. Take out the fetus from the uterus of pregnant sows at gestation day 35, wash with DPBS buffer solution containing 100U/mL penicillin and 100U/mL streptomycin.

2. Take the fetus obtained in step 1, use ophthalmic scissors to remove the head, limbs and viscera in the ultra-clean workbench, wash the remaining part with DPBS buffer, cut it into pieces with sterilized ophthalmic scissors, and transfer it to a 100mm petri dish. Cultivate in an incubator at 37°C, saturated humidity, and 5% CO ₂ for 4-6 hours.

3. After completing step 2, add DMEM culture solution containing 15% (volume percentage) fetal calf serum to the culture dish, and cultivate it in an incubator at 37°C, saturated humidity, and 5% _CO until the cells reach confluence Subculture when the degree is 90%. The culture medium used for subculture is DMEM culture medium containing 15% (volume percentage) fetal bovine serum.

4. Carry out follow-up experiments with pig fetal fibroblasts that have been subcultured to the third passage.

Embodiment 3, the effect of chaetocin on pig fetal fibroblasts

1. The pig fetal fibroblasts in the logarithmic growth phase prepared in Example 2 were inoculated into a 96-well plate ( ⁵ × 10 cells in each well), and cultivated in DMEM containing 15% (volume percentage) fetal bovine serum cultured at 37°C until the cells adhered to the wall.

2. After completing step 2, take the 96-well plate and perform the following grouping process:

Drug group 1: discard the culture supernatant, add DMEM culture solution containing 20nM chaetin (15% fetal bovine serum + 100U/mL ampicillin + 100U/mL streptomycin sulfate), and let stand at 37°C for 24 hours;

Drug group 2: discard the culture supernatant, add DMEM culture solution containing 40nM chaetin (15% fetal bovine serum + 100U/mL ampicillin + 100U/mL streptomycin sulfate), and let stand at 37°C for 24 hours;

Drug group 3: discard the culture supernatant, add DMEM culture solution containing 50nM chaetin (15% fetal bovine serum + 100U/mL ampicillin + 100U/mL streptomycin sulfate), and let stand at 37°C for 24 hours;

Drug group 4: discard the culture supernatant, add DMEM culture solution containing 100nM chaetin (15% fetal bovine serum + 100U/mL ampicillin + 100U/mL streptomycin sulfate), and let stand at 37°C for 24 hours;

Control group: the culture supernatant was discarded, and DMEM culture solution (15% fetal bovine serum + 0.1% double antibody) was added.

Cell viability was detected at 12h and 24h after treatment, respectively.

The result is shown in Figure 1. The results showed that chaetocin at a concentration higher than 40nM was more toxic to pig fetal fibroblasts. After 40nM chaetocin was used to treat porcine fetal fibroblasts for 24 hours, only about 50% of the cell viability remained.

Example 4, pig somatic cell nuclear transfer

1. Chaetin treatment of porcine fetal fibroblasts

1. Inoculate the pig fetal fibroblasts obtained in Example 2 into a ⁶ -well culture dish (1×10 cells per dish), adopt 37° C. of DMEM culture fluid containing 15% (volume percentage) fetal calf serum Culture statically until the cells grow to 90% confluence.

2. After completing step 2, take the petri dish and perform the following grouping:

Control group: discard the culture supernatant, add DMEM culture solution containing 0nM chaetin (15% fetal bovine serum + 100U/mL ampicillin + 100U/mL streptomycin sulfate), let stand at 37°C for 24 hours;

Drug group 1: discard the culture supernatant, add DMEM culture solution containing 10nM chaetin (15% fetal bovine serum + 100U/mL ampicillin + 100U/mL streptomycin sulfate), and let stand at 37°C for 24 hours;

Drug group 2: discard the culture supernatant, add DMEM culture solution containing 20nM chaetin (15% fetal bovine serum + 100U/mL ampicillin + 100U/mL streptomycin sulfate), let stand at 37°C for 24 hours;

Drug group 3: discard the culture supernatant, add 40nM chaetin-containing DMEM culture solution (15% fetal bovine serum+100U/mL ampicillin+100U/mL streptomycin sulfate), and stand at 37°C for 24 hours.

3. Take the COCs obtained in Example 1, digest them with 0.1% hyaluronidase to remove the cumulus cells, then wash them three times in IVM solution, and select mature oocytes with the first polar body and uniform and dense cytoplasm.

4. Take the "mature oocyte with the first polar body and uniform and dense cytoplasm" obtained in step 3, remove the first polar body and the surrounding cytoplasm, and then inject a nucleus donor cell (the nucleus donor cell is passed through step 2) The cultured porcine fetal fibroblasts) were processed in groups and located in the perivitelline space.

5. After completing step 4, move the oocytes injected with nuclei donor cells into the fusion tank, and use 150v/mm, 100μs, 2DC direct current pulses to fuse and activate the reconstructed embryos.

6. Put the reconstituted embryos obtained in step 3 into PZM-3 culture medium and culture them for 7 days (the culture conditions are: 39° C., saturated humidity, 5% CO ₂ ).

7. Observe and record the number of cleavages at the 48th hour of culture, and observe and record the number of cleavages and the number of blastocysts at the 7th day. The results are shown in Table 1.

Table 1 Statistical results of cleavage number and blastocyst number in different groups

Note: Statistically analyze the data of 3 repeated experiments, and calculate the mean ± standard error. All percentage data were subjected to t-test after arcsine transformation, and superscripts with different lowercase letters in the same column represent significant differences (P<0.05).

The results showed that the blastocyst rate on the 7th day was significantly higher than that of the control group and other drug groups when the porcine fetal fibroblasts (nuclear donor cells) were treated with 10nM chaetocin.

2. Treatment of reconstituted embryos with chaetin

1. Take the COCs cultured in Example 1, digest them with 0.1% hyaluronidase and remove the cumulus cells, then wash them three times in IVM solution, and select mature oocytes with the first polar body and uniform and dense cytoplasm.

2. Get the "mature oocyte with the first polar body and uniform and dense cytoplasm" obtained in step 1, remove the first polar body and the surrounding cytoplasm, and then inject a nucleus donor cell (the nucleus donor cell is the embodiment 2 obtained fetal fibroblasts) and localized them in the perivitelline space.

3. After completing step 2, move the oocytes injected with nuclei donor cells into the fusion tank, and use 150v/mm, 100μs, 2DC direct current pulses to fuse and activate the reconstructed embryos.

4. The reconstituted embryos obtained in step 3 were cultured according to the following groups (culture conditions: 39°C, saturated humidity, 5% CO ₂ ):

Control group: the reconstituted embryos were cultured in PZM-3 medium for 7 days.

Experimental group A: the reconstituted embryos were cultured in PZM-3 medium containing 0.5nM chaetin for 16 hours, and then transferred to PZM-3 medium for 7 days.

Experimental group B: the reconstituted embryos were cultured in PZM-3 medium containing 1.0 nM chaetin for 16 hours, and then transferred to PZM-3 medium for 7 days.

3. Observe and record the number of cleavages at the 48th hour of culture, and observe and record the number of cleavages and the number of blastocysts at the 7th day. The results are shown in Table 2.

Table 2 Statistical results of cleavage number and blastocyst number in different groups

Note: Statistically analyze the data of 4 repeated experiments, and calculate the mean ± standard error. All percentage data were subjected to t-test after arcsine transformation, and superscripts with different lowercase letters in the same column represent significant differences (P<0.05).

The results showed that the blastocyst rate on the 7th day was significantly higher than that of the control group and 1.0nM chaetocin group when the reconstituted embryos were treated with 0.5nM chaetocin.

Claims (2)

1. A method for animal somatic cell nuclear transplantation, comprising the steps of: cultivating the donor cell with a nuclei-donating cell culture fluid; containing 10nM chaetin in the nuclei-donating cell culture fluid; Elements and basic cell culture fluid; The basic cell culture fluid is DMEM culture fluid; The nuclear donor cells are pig fetal fibroblasts; The methods are non-disease treatment methods. 2. A method for animal somatic cell nuclear transfer, characterized in that: the method comprises two stages of cultivation; the first stage, cultivating reconstituted embryos with reconstituted embryo culture fluid; Reconstructed embryos were cultivated in the culture medium; the culture medium of the reconstituted embryos contained 0.5nM chaetocin; The first stage is 16h; The preparation method of the reconstructed embryo is as follows: injecting a nucleus-donating cell into the perivitelline space of the enucleated oocyte, electric shock fusion and activation of the reconstructed embryo; The conditions of the electric shock fusion are: a direct current pulse of 150v/mm, 100 µs, 2DC; The preparation method of the "enucleated oocyte" is: remove the first polar body and nucleus of the porcine mature oocyte by blind aspiration; The method for preparing porcine mature oocytes comprises the following steps: (c1)-(c3): (c1) Take the isolated porcine ovary, extract the follicular fluid in the follicle, and select the cumulus-oocyte complexes (COCs) wrapped with 3-5 layers of cumulus cells; (c2) Transfer the cumulus-oocyte complexes (COCs) obtained in step (c1) to IVM solution, and culture them in an incubator at 39°C, saturated humidity, and 5% CO ₂ for 42 hours; (c3) After completing step (c2), remove the cumulus cells after digestion with 0.1% hyaluronidase, and select mature oocytes with the first polar body and uniform and dense cytoplasm; The IVM solution is composed of a solvent and a solute, and the solute and its concentration in the IVM solution are: 199 medium 0.987g/100mL, follicular fluid 10mL/100mL, epidermal growth factor 1 μg/100mL, follicle-stimulating hormone 50mg/100mL , Luteinizing hormone 50mg/100mL, cysteine 7μg/100mL; The solvent is pure water; The reconstituted embryo culture fluid includes chaetocin and basic embryo culture fluid; the basic embryo culture fluid is PZM-3 culture fluid; The methods are non-disease treatment methods.