Method for improving efficiency of pig somatic cell nuclear transfer

Abstract

The application belongs to the technical field of animal somatic cell nuclear transfer, and provides a method for improving the efficiency of pig somatic cell nuclear transfer, which improves the efficiency of pig somatic cell nuclear transfer by using small molecule compounds and traditional Chinese medicine monomers to treat differentiated donor cells, oocytes and somatic cell nuclear transfer embryos in time sequence. The application uses traditional Chinese medicine monomers to treat pig oocytes and somatic cell nuclear transfer embryos in time sequence, which can effectively reduce the stress damage caused by the external environment, solves the problems of long culture cycle of oocytes and somatic cell nuclear transfer embryos, and is not conducive to large-scale production. Compared with artificially synthesized antioxidant substances, traditional Chinese medicine monomers have higher biological activity and structural diversity, are more likely to enter oocytes and somatic cell nuclear transfer embryos to play a pharmacological effect, have higher biological activity, and have industrialization prospects.

Description

Technical Field

[0001] This invention relates to the field of animal somatic cell nuclear transfer technology, specifically a method for improving the efficiency of porcine somatic cell nuclear transfer. Background Technology

[0002] Somatic cell nuclear transfer (SCNT) is an asexual reproduction technique that reprograms the nucleus of terminally differentiated somatic cells into a totipotent state, producing offspring with the same genotype as the donor cells. Due to its unique technological advantages, SCNT has been widely applied in many related fields. For example, it can be used to rapidly breed animal varieties with superior qualities; it can be used to mass-produce transgenic animals, thereby significantly increasing production while reducing costs; and it can be used to clone organs for organ transplantation. Therefore, this technology has broad application prospects in breeding superior livestock, treating human genetic diseases, and protecting rare and endangered wild animals. Since the birth of Dolly the sheep in 1997, the technology has been successfully applied to other mammals such as pigs, cattle, horses, camels, and monkeys. However, the efficiency of this technology remains very low. For example, the birth rate of cloned pigs is only 1-2% (number of births / number of eggs used), the stillbirth rate is high (20-30%), and the malformation rate is high (30-50%). These problems have significantly restricted the development of research on somatic cell cloned animals and transgenic cloned animals.

[0003] Donor cells are the starting point for somatic cell nuclear transfer (SCLT) embryo development, and their epigenetic modification state directly affects the developmental capacity of SCLT embryos. Currently, incomplete or incorrect reprogramming of the donor cell nucleus is considered the main reason for low SCLT efficiency. Numerous studies have found significant differences in SCLT efficiency among different types of donor cells, with donor cells exhibiting higher acetylation and low methylation levels showing higher SCLT efficiency. Furthermore, research has shown that during oocyte maturation and SCLT embryo development, the culture environment or accumulation of metabolic products can lead to oxidative stress, resulting in abnormal oocyte and SCLT embryo development.

[0004] Therefore, in view of the above situation, there is an urgent need to provide methods to improve the efficiency of porcine somatic cell nuclear transfer in order to overcome the shortcomings in current practical applications. Summary of the Invention

[0005] The purpose of this invention is to provide a method for improving the efficiency of porcine somatic cell nuclear transfer, thereby addressing the problems mentioned in the background art.

[0006] This invention is achieved by using a method to improve the efficiency of porcine somatic cell nuclear transfer. The method improves the efficiency of porcine somatic cell nuclear transfer by using a combination of small molecule compounds and traditional Chinese medicine monomers to process differentiated donor cells, oocytes and somatic cell nuclear transfer embryos in a sequential manner.

[0007] As a further aspect of the present invention: the combination of small molecule compounds and traditional Chinese medicine monomers includes the following components: PDK activator, mangiferin, SHP-141, GSK3685032 and tanshinone IIA.

[0008] As a further aspect of the present invention: the combination of small molecule compounds and traditional Chinese medicine monomers is used in stages according to time sequence, including: first stage compound (donor cell culture stage), second stage traditional Chinese medicine monomer (oocyte maturation stage), third stage compound (within 72 hours of reconstructed embryo culture) and fourth stage traditional Chinese medicine monomer (after 72 hours to blastocyst stage);

[0009] Phase 1: Donor cells were cultured in a cell culture medium containing PDK activator; the medium was based on DMEM and supplemented with 0-100 μM PDK activator (PS48), 10% FBS and 1% penicillin and streptomycin.

[0010] Phase 2: Porcine oocytes were cultured in vitro using a porcine oocyte maturation medium containing mangiferin. The medium was based on TCM-199 and supplemented with 25-100 μM mangiferin, 10% porcine follicular fluid, 10 IU / mL eCG, 10 IU / mL hCG, 0.91 mmol / L sodium pyruvate, 10 ng / mL EGF, and 1% penicillin and streptomycin.

[0011] The third stage: porcine somatic cell nuclear transfer embryos were cultured in an in vitro culture medium containing SHP-141 and GSK3685032 to form reconstructed embryos; the culture medium was based on PZM-3 and supplemented with 0-100 nM SHP-141 and 0-100 nM GSK3685032.

[0012] Fourth stage: Porcine somatic cell nuclear transfer embryos are cultured in an in vitro culture medium containing tanshinone IIA to form blastocysts; the culture medium is based on PZM-3 and supplemented with 0-100 μM tanshinone IIA.

[0013] As a further aspect of the present invention: the small molecule compounds and traditional Chinese medicine monomers must be brought into contact with the corresponding donor cells, oocytes or somatic cell nuclear transfer embryos in a certain sequence.

[0014] The combination of small molecule compounds and traditional Chinese medicine monomers provided by this invention can improve the efficiency of other somatic cell nuclear transplantation animals by adjusting their components, concentrations and action sequences, and this also falls within the application scope of the combination of small molecule compounds and traditional Chinese medicine monomers.

[0015] The mechanism of this invention is as follows: This invention provides a combination of small molecule compounds and traditional Chinese medicine monomers that can improve the efficiency of porcine somatic cell nuclear transfer, and further provides a combination of small molecule compounds and traditional Chinese medicine monomers for use in stages according to a time sequence. The treatment of the small molecule compounds puts the donor cells at a high level of acetylation and low level of methylation, thereby improving the accuracy and efficiency of donor cell reprogramming; the treatment of the traditional Chinese medicine monomers establishes and maintains the hypoxic environment required for oocyte and embryo development to antagonize the adverse effects of oxidative stress; by utilizing the relay combination of small molecule compounds and traditional Chinese medicine monomers, the in vivo and in vitro development efficiency of porcine somatic cell nuclear transfer embryos is improved.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] By using combinations of small molecule compounds to process donor cells in a time-sequential, phased manner, the accuracy and reliability of reprogramming can be improved, while also facilitating precise quality control and standardized operation.

[0018] By using a combination of traditional Chinese medicine monomers to treat porcine oocytes and somatic cell nuclear transfer embryos in stages according to a specific time sequence, stress damage caused by the external environment can be effectively reduced. This solves the problems of long culture cycles for oocytes and somatic cell nuclear transfer embryos and the difficulty in large-scale production. Compared with artificially synthesized antioxidants, the combination of traditional Chinese medicine monomers has higher biological activity and structural diversity, and is more likely to enter oocytes and somatic cell nuclear transfer embryos to exert its effects. It has higher biological activity and has industrialization prospects. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the effect of PDK activators on the proliferation activity of donor cells in vitro.

[0020] Figure 2 This is a schematic diagram illustrating the effect of PDK activators on the activity of deacetylases in donor cells in vitro.

[0021] Figure 3 This is a schematic diagram illustrating the effect of mangiferin on the ROS level in mature porcine oocytes in vitro. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0023] This invention utilizes a combination of small molecule compounds and traditional Chinese medicine monomers to sequentially process differentiated donor cells, oocytes, and somatic cell nuclear transfer embryos, thereby improving the efficiency of porcine somatic cell nuclear transfer.

[0024] Example 1

[0025] 1. Isolation and culture of porcine fetal fibroblasts:

[0026] The uterus was aseptically removed from the sow at 33-35 days of gestation (with both ends of the uterus tied tightly with sterile gauze), rinsed 2-3 times with physiological saline containing 100 IU / mL penicillin and 100 μg / mL streptomycin, and then placed in a 4℃ foam box and sealed for 30 minutes before being transported to the laboratory.

[0027] In a clean bench, the fetus is aseptically removed and rinsed with physiological saline containing 100 IU / mL penicillin and 100 μg / mL streptomycin. The head, limbs and internal organs are removed with ophthalmic scissors. The remaining part is rinsed with the above liquid, cut into small pieces and spread on the bottom of a cell culture dish. After being placed in an incubator at 38°C with 5% CO2 for 4-6 hours, cell culture medium is slowly added, while avoiding floating of the tissue pieces. The cells are then cultured in an incubator at 38°C with 5% CO2 until the cell confluence reaches 90%, at which point the cells are passaged.

[0028] Subsequent experiments were conducted using porcine fetal fibroblasts passaged to the 3rd-5th generation.

[0029] 2. Regulation of porcine fetal fibroblasts (first-stage compound):

[0030] The aforementioned logarithmically growing porcine fetal fibroblasts were seeded into 96-well plates (4-5 × 10⁶ cells / well). 4 ), 12-hole plate (5×10) 5 ) and 6-hole plate (1.2×10 6 The cells were cultured in DMEM medium containing 15% fetal bovine serum at 38°C until they adhered to the culture wall. After the above treatment, the medium was replaced with the first-stage medium after 12 hours and cultured for 24-48 hours.

[0031] The first stage culture medium was based on DMEM and supplemented with 0-100 μM PDK activator (PS48), 10% FBS and 1% penicillin and streptomycin.

[0032] The experimental results and analysis are as follows:

[0033] 1. Effects of PDK activators on in vitro donor cell proliferation efficiency:

[0034] Comparative Example 1 (0 μM) served as the blank control group, while experimental groups were prepared with different doses of PDK activator (2.5-50 μM). The effects of different doses of PDK activator on the in vitro donor cell proliferation efficiency were observed in each example. The results are as follows: Figure 1 As shown, adding 7.5 μM PDK activator to the donor cell culture medium resulted in significantly higher donor cell proliferation efficiency within 24-72 h compared to the control and other concentration treatment groups.

[0035] 2. Effects of PDK activators on deacetylase activity in in vitro donor cells:

[0036] Comparative Example 1 (0 μM) served as the blank control group, while experimental groups were prepared with different doses of PDK activator (2.5-50 μM). The effects of different doses of PDK activator on the in vitro donor cell proliferation efficiency were observed in each example. The results are as follows: Figure 2 As shown, compared with the control group, the addition of 7.5 μMPDK activator to the donor cell culture medium significantly reduced the intracellular histone deacetylase (HDAC) activity.

[0037] Example 2

[0038] 1. Ovarian collection and transportation:

[0039] Pig ovaries were obtained from slaughterhouses near the city, then placed in thermos flasks containing 1% penicillin and physiological saline at 33-35°C, and returned to the laboratory within 4 hours.

[0040] 2. Oocyte separation:

[0041] Use a 20mL syringe to draw 3-5mm of follicular fluid from the surface of the ovary. Place the follicular fluid in a test tube in a 38.5℃ constant temperature rack, let it stand for 15-20 minutes, discard the supernatant, and aliquot the precipitate with basal culture medium TCM-199.

[0042] Select cumulus-oocyte complexes containing more than three layers of cumulus cells and good refractive properties under a stereomicroscope.

[0043] 3. In vitro maturation of oocytes (second stage of traditional Chinese medicine monomers):

[0044] After the treatment in step 2 above, the cumulus-oocyte complex was placed in preheated porcine oocyte in vitro maturation culture medium and then cultured in an environment with a CO2 volume concentration of 5% and a temperature of 38.5°C for 40-44 hours.

[0045] The second-stage culture medium was based on TCM-199 and supplemented with 0-100 μM mangiferin, 10% porcine follicular fluid, 10 IU / mL eCG, 10 IU / mL hCG, 0.91 mol / L sodium pyruvate, 10 ng / mL EGF and 1% penicillin and streptomycin.

[0046] The experimental results and analysis are as follows:

[0047] 1. Effects of mangiferin on ROS levels in mature porcine oocytes in vitro:

[0048] Comparative Example 1 (0 μM) served as the blank control group, while experimental groups were prepared with different doses of mangiferin. The effects of different doses of mangiferin on ROS activity in porcine mature oocytes were observed. Results are as follows: Figure 3 As shown, mangiferin can significantly reduce the level of ROS in mature oocytes, with the group that added 25-50 μM mangiferin showing the best effect, indicating that mangiferin has a good protective effect on the in vitro maturation of porcine oocytes.

[0049] 2. Effects of mangiferin on in vitro maturation rate of porcine oocytes and parthenogenetic embryo development:

[0050] Comparative Example 1 (0 μM) served as the blank control group, while the experimental groups were those with different doses of mangiferin. The effects of different doses of mangiferin on porcine oocyte maturation and early parthenogenetic embryo development were observed. The results are shown in Table 1. Mangiferin had no significant effect on the in vitro maturation rate of porcine oocytes, but it significantly improved the early embryonic development capacity. Among them, the effect of adding 50 μM mangiferin on early porcine embryonic development was the best.

[0051] Table 1. Effects of mangiferin on in vitro maturation rate of porcine oocytes and parthenogenetic embryo development.

[0052]

[0053] Example 3

[0054] 1. The enucleation process for oocytes is as follows:

[0055] Mature oocytes with uniform cytoplasm, intact plasma membranes, and extruded polar bodies were selected and simultaneously transferred into the manipulation solution (TCM-199 and 2.5 μg / mL cytochalasin B) along with nuclear donor cells. The oocytes were then enucleated under a micromanipulation system.

[0056] 2. The steps for injecting donor cells are as follows:

[0057] Under a microsurgical system, a moderately sized, smooth-surfaced fetal fibroblast is aspirated using an injection needle and injected into the periovarian space through the enucleation incision of the oocyte. The zona pellucida is then gently pressed to bring the donor cell into contact with the recipient oocyte's plasma membrane, facilitating fusion.

[0058] 3. The fusion / electroactivation steps are as follows:

[0059] The reconstructed embryo was washed three times in an activation solution (PAF) containing 5 μg / mL CB and 10 μg / mL CHX of PZM-3, equilibrated for 3 min, and then quickly transferred to a fusion tank for fusion / electroactivation.

[0060] 4. The steps for reconstructing embryo culture (stage three compounds) are as follows:

[0061] The activated reconstructed embryos were washed three times with PZM-3 and then transferred to a liquid in PZM-3 that had been pre-equilibrated in a CO2 incubator for at least 4 hours and covered with paraffin oil for 7 days. During this period, the development of cleavage, morula and blastocyst was checked and recorded.

[0062] The third-stage culture medium was based on PZM-3 with the addition of 0-100nM SHP-141 and 0-100nM MSK3685032, and cultured for 24 hours.

[0063] The experimental results and analysis are as follows:

[0064] 1. Effects of SHP-141 treatment on reconstructed embryos on early embryonic development:

[0065] Comparative Example 1 (0 nM) served as the blank control group, while the experimental groups were those supplemented with different doses of SHP-141. The effects of different doses of SHP-141 on early embryonic development were observed. The results are shown in Table 2. SHP-141 can significantly improve the developmental capacity of early embryos, with the best effect observed in the addition of 50 nM SHP-141 on the early development of reconstructed embryos.

[0066] 2. Effects of GSK3685032 treatment on reconstructed embryos on early embryonic development:

[0067] Comparative Example 1 (0 nM) served as the blank control group, while the experimental groups were those supplemented with different doses of GSK3685032. The effects of different doses of GSK3685032 on early embryonic development were observed. The results are shown in Table 3. GSK3685032 significantly improved the early embryonic development capacity, with the best effect observed in the addition of 75 nM GSK3685032 on the early development of reconstructed embryos.

[0068] 3. Effects of combined treatment of reconstructed embryos with SHP-141 and GSK3685032 on early embryonic development:

[0069] Comparative Example 1 (0 nM) served as the blank control group, while the experimental group consisted of both SHP-141 and GSK3685032. The effects of different dosage combinations on early embryonic development were observed. The results are shown in Table 4. The combined application of SHP-141 and GSK3685032 significantly improved the early embryonic development capacity, with the addition of SHP-141 (50 nM) and GSK3685032 (50 nM) showing the best effect on the early development of reconstructed embryos.

[0070] Table 2. Effects of different concentrations of SHP-141 on the development of reconstructed embryos after 24 hours.

[0071]

[0072] Table 3. Effects of different concentrations of GSK3685032 on the development of reconstructed embryos after 24 hours.

[0073]

[0074]

[0075] Table 4. Effects of combined treatment of SHP-141 and GSK3685032 on early embryonic development in reconstructed embryos.

[0076]

[0077] Example 4

[0078] 1. The oocyte enucleation, donor cell injection, and fusion / electroactivation steps are the same as in Example 3;

[0079] 2. Improving the developmental efficiency of porcine somatic cell nuclear transfer embryos (fourth-stage traditional Chinese medicine monomers):

[0080] The reconstructed embryos obtained in Example 3 (after 72 hours of in vitro culture) were cultured for 4 days with 0-100 μM tanshinone IIA added to the original solution and placed in an environment with a CO2 volume concentration of 5% and a temperature of 38.5°C. Then, the blastocyst development rate and blastocyst cell number were detected.

[0081] The experimental results and analysis are as follows:

[0082] 1. Effects of tanshinone IIA on mid-to-late stage embryonic development:

[0083] Comparative Example 1 (0 μM) served as the blank control group, while the experimental groups were treated with different doses of tanshinone IIA. The effects of different doses of tanshinone IIA on mid-to-late stage embryonic development were observed. The results are shown in Table 5. Tanshinone IIA significantly improved early embryonic development, with the best effect observed in mid-to-late stage embryonic development when 40 μM tanshinone IIA was added.

[0084] Table 5. Effects of different doses of tanshinone IIA on mid-to-late-stage embryonic development.

[0085]

[0086]

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for improving the efficiency of porcine somatic cell nuclear transfer, characterized in that, The method improves the efficiency of porcine somatic cell nuclear transfer by using a combination of small molecule compounds and traditional Chinese medicine monomers to treat differentiated donor cells, oocytes and somatic cell nuclear transfer embryos in a sequential manner. The combination of small molecule compounds and traditional Chinese medicine monomers includes PDK activator, mangiferin, SHP-141, GSK3685032, and tanshinone IIA; The combination of small molecule compounds and traditional Chinese medicine monomers is used in stages according to a time sequence, including: Phase 1: Donor cells were cultured in a cell culture medium containing a PDK activator at a concentration of 7.5 μM. Second stage: Porcine oocytes were cultured in vitro using a porcine oocyte maturation culture medium containing mangiferin, wherein the concentration of mangiferin was 50-100 μM; The third stage: porcine somatic cell nuclear transfer embryos were cultured in an in vitro culture medium containing SHP-141 and GSK3685032 to form reconstructed embryos. The concentration of SHP-141 was 50 nM and the concentration of GSK3685032 was 75 nM. Fourth stage: Porcine somatic cell nuclear transfer embryos are cultured in an in vitro culture medium containing tanshinone IIA to form blastocysts, wherein the concentration of tanshinone IIA is 20-80 μM.

2. The method for improving the efficiency of porcine somatic cell nuclear transfer according to claim 1, characterized in that, In the first stage, the culture medium was based on DMEM and supplemented with 7.5 μM PDK activator, 10% FBS and 1% penicillin and streptomycin.

3. The method for improving the efficiency of porcine somatic cell nuclear transfer according to claim 1, characterized in that, In the second stage, the culture medium was based on TCM-199 and supplemented with 50 μM mangiferin, 10% porcine follicular fluid, 10 IU / mL LeCG, 10 IU / mL hCG, 0.91 mmol / L sodium pyruvate, 10 ng / mL EGF and 1% penicillin and streptomycin.

4. The method for improving the efficiency of porcine somatic cell nuclear transfer according to claim 1, characterized in that, In the third stage, the culture medium was based on PZM-3 and supplemented with 50 nM SHP-141 and 75 nM S SK3685032.

5. The method for improving the efficiency of porcine somatic cell nuclear transfer according to claim 1, characterized in that, In the fourth stage, the culture medium was based on PZM-3 and supplemented with 40 μM tanshinone IIA.

6. The method for improving the efficiency of porcine somatic cell nuclear transfer according to claim 1, characterized in that, The small molecule compounds and traditional Chinese medicine monomers are contacted with the corresponding donor cells, oocytes, or somatic cell nuclear transfer embryos in a predetermined sequence.

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