CN113174410A - Method for improving the means of donor-acceptor cell adhesion in manual cloning procedures - Google Patents
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Abstract
The invention discloses an improved method for bonding donor-acceptor cells in a manual cloning procedure, which comprises the steps of bonding enucleated half eggs and donor cells by Phytohemagglutinin (PHA) to form half egg-donor cell coupled bodies; then the other enucleated half egg is bonded with a counterpart to form an egg-donor-egg type bonded body (forming a triangle shape), the bonded body is transferred into an electric fusion liquid to be fused, fusion is carried out in an egg-donor-egg bonding mode, and the fusion efficiency is obviously higher than that in the egg-donor bonding mode.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of cloning, in particular to an improved method for a donor-acceptor cell adhesion mode in a manual cloning procedure.
[ background of the invention ]
Animal cloning refers to a process of directly obtaining offspring with the same genetic character as a parent without sexual reproduction of an animal, and comprises parthenogenesis, blastomere separation and culture, embryo segmentation, nuclear transfer and the like. At present, the widely known animal cloning technique is actually referred to as a nuclear transfer technique. The traditional cell nucleus transplantation technology generally refers to a method of micromanipulation, wherein a cell nucleus of a donor cell is injected into a mature oocyte or an early zygote which is pre-enucleated to form a new reconstructed embryo, the reconstructed embryo is subjected to fusion activation and then is subjected to development programming and reprogramming, and a new individual is developed in a surrogate mother body through cell division and differentiation, wherein the complex processes of a series of operations including oocyte enucleation, donor cell injection, donor cell fusion and activation, in vitro culture, embryo transplantation and the like can influence the final efficiency of the nuclear transplantation. Researchers have been constantly improving each and every technology step in the nuclear transfer process since the birth of the "Dolly" sheep in 1997.
The manual cloning (HMC) technique improves the denucleation operation by directly cutting the denucleation with a special cutting knife under a stereomicroscope, then bringing two half eggs without cell nucleus close to a donor cell and applying a direct current pulse to fuse the two half eggs, and finally culturing the reconstructed embryo individually. The whole process does not require a micromanipulator, which is called manual cloning by Vajta et al (Vajta et al, 2001). Research on Duyutao (2011) shows that compared with traditional somatic cell nuclear transplantation, the manual cloning technology can greatly improve the blastocyst rate of the somatic cell nuclear transplantation.
The basic procedure of manual cloning includes: mature culture of the oocyte; culturing fetal fibroblasts; manual enucleation of oocytes; for-binding/fusion/activation of recipient cells; micro-hole culture of the reconstructed embryos. The nucleus is removed from the oocyte cytoplasm and the process is called enucleation. The denucleation of the oocyte is a key technical link of somatic cell nuclear transfer technology, and the denucleation is ensured to be clean under the condition of removing cytoplasm as little as possible. If the cytoplasm is removed too much, proteins and mRNAs accumulated in the oocyte are lost too much, and reprogramming of the donor cell is affected. If the denucleation is incomplete, chromosome aneuploidy of the reconstructed embryo can be caused, polyploidy, abnormal cleavage, stunting, early death of the embryo and the like are caused, and the denucleation efficiency is directly related to the in vitro development of the constructed cloned embryo and the pregnancy rate of receptor transplantation and the calving rate.
The traditional nuclear transplantation techniques include a blind enucleation method, a staining enucleation method and a spin-view method, but each method has certain defects, and people always seek better methods. Such as (Liudong, 2004), the oocyte is pushed through a notch in the zona pellucida adjacent the polar body with a glass needle, causing the first polar body, with 1/4-1/3 cytoplasm adjacent thereto, to exit the zona pellucida. (Guo Tong, 2002) bovine oocytes were cultured in a short time in a hypertonic solution of sucrose, with the spindle portion of most of the oocytes protruding above the surface of the egg membrane, indicating an enucleation operation. These have attempted to improve methods for enucleating oocytes. Later developed manual cloning techniques (Vajta 2007) directly cut half of the eggs with a knife to achieve the goal of denucleation clean.
The blind enucleation method is characterized in that according to the characteristics of mature oocytes, about 1/3 cytoplasm near a polar body is removed, the enucleation purpose is achieved, the operation is simple and rapid, but the position of the first polar body of the oocytes is changed in the enucleation process, the enucleation effect is influenced, the enucleation efficiency is relatively low, and generally 70 percent of enucleation efficiency is achieved. Staining enucleation after staining the entire oocyte with the DNA-specific dye Hoechest33342, a method of revealing chromosomal enucleation by fluorescence microscopy can remove a small amount of cytoplasm and ensure 100% clean, but ultraviolet irradiation damages embryo viability (Tsunda, 1988).
The spindle-view method developed later is to utilize polarized light refraction to directly obtain a spindle mirror image, and the spindle in the oocyte can be observed without dyeing, the denucleation rate can reach more than 95%, but the price is high, and the spindle-view method is not beneficial to popularization and application. The enucleation is directly performed by cutting with a knife, the number of the removed cytoplasm is more, and then 2 half eggs are fused to make up for the loss of excessive cytoplasm removal.
Donor-recipient cell fusion in conventional nuclear transfer procedures is achieved by injecting donor cytoplasm into enucleated oocytes through a micromanipulator, followed by electrically/chemically activated donor-recipient cell fusion.
[ summary of the invention ]
In the manual cloning procedure in the prior art, the oocyte is removed from the zona pellucida, donor cells are fused into the oocyte, and donor-acceptor cells can only be firstly bonded together, and then the donor-acceptor cells are fused in an electric activation/chemical activation mode.
The invention relates to a method for improving the binding mode of donor-acceptor cells in a manual cloning procedure, which comprises the following steps:
1) adhering enucleated clean oocytes to donor cells:
digesting donor cells by pancreatin, immediately stopping the donor cells after the donor cells become round, digesting 50-60 donor cells each time, putting the donor cells into a culture dish after the donor cells become round, wherein the liquid in the culture dish is oocyte culture solution containing Phytohemagglutinin (PHA) of 0.3-0.5mg/mL, and sparsely arranging the oocyte culture solution to wait for adhesion;
transferring the enucleated and clean oocyte 20-40 times to the culture dish of the first step each time, and stirring the enucleated and clean oocyte by using a pointed glass tube to be adhered with the donor cell obtained in the first step to form a half egg-donor cell pairing body;
2) binding another enucleated clean oocyte to the donor-recipient cell counterpart:
thirdly, shifting the other denucleated oocyte along the gap of the half egg-donor cell pairing body obtained in the second step by using a pointed glass tube, and bonding the denucleated oocyte and the half egg-donor cell pairing body to form a planar triangular oocyte-somatic cell-oocyte pairing body (short for egg-donor-egg) with the donor cell in the middle and the denucleated oocyte on two sides;
transferring the oocyte-somatic cell-oocyte coupled body obtained in the step (III) into oocyte culture solution, bonding 15-25 oocyte-somatic cell-oocyte coupled bodies each time, transferring into electric fusion solution to be fused, completely fusing donor cells and acceptor cells into a whole, and facilitating the starting of the subsequent nuclear mass reprogramming program.
In the invention:
the donor cell in the step 1) is a porcine fetal fibroblast.
Digesting with pancreatin in the step 1), wherein the concentration of the pancreatin is 0.25 percent, and the digestion time is 3-5 min.
The culture dish in the step 1) is selected from a 60mm culture dish.
The oocyte culture solution of the step 1) and the step 2) comprises 80% by volume of TCM199, 10% by volume of pig follicular fluid, 10% by volume of fetal bovine serum, 0.1g/L cysteine, 0.075g/L penicillin, 0.05g/L streptomycin, 10ng/mL insulin-like growth factor, 50ng/mL epidermal growth factor, 2.2g/LNaHCO310IU/mL pregnant mare serum gonadotropin, 10IU/mL human chorionic gonadotropin and 10-30 mu mol/L butylbenzoic acid; the main components of the electric activation liquid are: 0.3mmol/L mannitol, 0.1mmol/L CaCl2·H2O、0.1mmol/L MgSO4·7H2O, 0.5mmol/L Hepes, 0.01% (w/v) polyvinyl alcohol (PVA).
The oocyte culture solution of step 1) contains Phytohemagglutinin (PHA) in an amount of 0.4 mg/mL.
The gap of the half egg-donor cell pairing body in the step 2) refers to the bonding position of the enucleated oocyte and the donor cell.
Moving the bonded oocyte-somatic cell-oocyte paired body into a fusion groove for fusion-electric activation, applying an alternating electric field (alternating current pulse of 1-3V/mm), enabling the oocyte-somatic cell-oocyte paired body to automatically lean against an electrode, wherein an enucleated oocyte of one pole is tightly attached to one end of the electrode, an oocyte of the other pole is suspended in the fusion groove, a somatic cell is clamped between two enucleated oocytes, and the oocyte-somatic cell-oocyte paired body is perpendicular to the magnetic field of the electrode, and the electric fusion parameter is AC: 6V/cm; DC: 1.2kV/cm, 30 mu s, 2DC, after electrically activated reconstructed embryo is transferred into embryo culture solution and washed for 3 times, the reconstructed embryo is recovered in an incubator at 38.5 ℃ for 30min, and then the fusion condition is checked.
Compared with the prior art, the invention has the following advantages:
1. whether the cytoplasm of the enucleated pig oocyte can be successfully fused with a donor cell is a key step influencing the cloning efficiency, and in the manual cloning process, because a part of cytoplasm is lost due to the cutting and enucleation, an enucleated oocyte needs to be taken to be fused with the donor cell in the fusion process with the donor cell so as to improve the reprogramming efficiency of nuclear transfer. The method for improving the adhesion mode of the donor-acceptor cells in the manual cloning procedure is an adhesion mode for improving the fusion efficiency of the donor-acceptor cells, and through comparative experiments, the method for improving the adhesion mode of the donor-acceptor cells in the manual cloning procedure disclosed by the invention has the advantages that the fusion is carried out in an egg-donor-egg adhesion mode, the efficiency is up to 88.03 +/-0.58, and the fusion is carried out in an egg-donor-egg adhesion mode, and the efficiency is 79.95% +/-0.82. The former fusion efficiency was significantly higher than the latter.
2. The adhesion mode of the hemicytoplasm after denucleation and the donor plays a key role in the fusion of the donor and the acceptor, and the improved method of the adhesion mode of the donor and the acceptor cells in the manual cloning procedure adopts the adhesion method of the ovum-donor type in the prior art, thereby being simple and convenient for operation, but being relatively time-consuming and low in fusion efficiency; the egg-donor-egg type bonding method adopting the method of the invention has the advantages of less time consumption and high fusion efficiency, and compared with the time for bonding a matched body, the egg-donor-egg type bonding method only needs 23s, while the egg-donor-egg type bonding method needs 30 s. In the improved method for the adhesion mode of the donor-acceptor cells in the manual cloning procedure, the fusion rate of the egg-donor-egg type is obviously higher than 88.03% vs 79.95% of the egg-donor type in the comparative example 1, so that the adhesion mode of the donor-acceptor cells in the improved method for the manual cloning procedure is feasible, and is worthy of popularization and application.
[ description of the drawings ]
FIG. 1 is a diagram showing the manner of egg-donor-egg adhesion according to example 1 of the present invention.
FIG. 2 is a view of a pointed glass tube (head in a blunt round shape) used in example 1 of the present invention.
Fig. 3 is a graph showing the egg-donor adhesion pattern of comparative example 1 of the present invention.
[ detailed description ] embodiments
The following examples are provided to further illustrate the embodiments of the present invention.
Example 1:
a method for improving the means of donor-recipient cell adhesion in a manual cloning procedure, comprising the steps of:
1) adhering enucleated clean oocytes to donor cells (porcine fetal fibroblasts):
digesting donor cells by pancreatin, wherein the pancreatin concentration is 0.25%, digesting for 4min, stopping immediately after the donor cells are rounded, digesting 60 donor cells each time, placing the donor cells in a 60mm culture dish (the culture dish is used for replacing the culture dish) after the donor cells are rounded, and allowing the liquid in the culture dish to be oocyte culture solution containing Phytohemagglutinin (PHA) of 0.4mg/mL, sparsely arranging the oocyte culture solution and waiting for adhesion;
transferring 30 enucleated oocytes to the culture dish of the first step each time, and stirring the enucleated oocytes by using a pointed glass tube to be adhered with the donor cells obtained in the first step to form a half egg-donor cell pairing body;
2) binding another enucleated clean oocyte to the donor-recipient cell counterpart:
thirdly, the other enucleated oocyte is stirred by a pointed glass tube along the gap of the half egg-donor cell pairing body (the bonding position of the enucleated oocyte and the donor cell) obtained in the second step and is bonded with the half egg-donor cell pairing body to form a plane triangle-shaped oocyte-somatic cell-oocyte pairing body (egg-donor-egg for short) with the donor cell in the middle and the enucleated oocyte at two sides;
transferring the oocyte-somatic cell-oocyte coupled body obtained in the step (III) into oocyte culture solution, bonding 15-25 oocyte-somatic cell-oocyte coupled bodies each time, transferring into electric fusion solution to be fused, so that donor cells and acceptor cells are completely fused into a whole, and the subsequent starting of a nuclear mass reprogramming program is facilitated;
the oocyte culture solution of the step 1) and the step 2) comprises 80% by volume of TCM199, 10% by volume of pig follicular fluid, 10% by volume of fetal bovine serum, 0.1g/L cysteine, 0.075g/L penicillin, 0.05g/L streptomycin, 10ng/mL insulin-like growth factor, 50ng/mL epidermal growth factor, 2.2g/LNaHCO310IU/mL pregnant mare serum gonadotropin, 10IU/mL human chorionic gonadotropin and 10-30 mu mol/L butylbenzoic acid; the main components of the electric activation liquid are: 0.3mmol/L mannitol, 0.1mmol/L CaCl2·H2O、0.1mmol/L MgSO4·7H2O, 0.5mmol/L Hepes, 0.01% (w/v) polyvinyl alcohol (PVA);
and 2) moving the bonded oocyte-somatic cell-oocyte paired body into a fusion groove for fusion-electric activation, applying an alternating electric field (alternating current pulse of 1-3V/mm), and enabling the oocyte-somatic cell-oocyte paired body to automatically lean against an electrode, wherein the enucleated oocyte of one pole is tightly attached to one end of the electrode, the oocyte of the other pole is suspended in the fusion groove, and the somatic cell is clamped between the two enucleated oocytes, so that the oocyte-somatic cell-oocyte paired body is perpendicular to the magnetic field of the electrode, and the electric fusion parameter AC: 6V/cm; DC: 1.2kV/cm, 30 mu s, 2DC, after electrically activated reconstructed embryo is transferred into embryo culture solution and washed for 3 times, the reconstructed embryo is recovered in an incubator at 38.5 ℃ for 30min, and then the fusion condition is checked.
Example 2:
a method for improving the means of donor-recipient cell adhesion in a manual cloning procedure, comprising the steps of:
1) adhering enucleated clean oocytes to donor cells (porcine fetal fibroblasts):
digesting donor cells by pancreatin, wherein the pancreatin concentration is 0.25%, digesting for 3min, stopping immediately after the donor cells are rounded, digesting 50 donor cells each time, placing the donor cells in a 60mm culture dish (the culture dish is used for replacing the donor cells), and enabling the liquid in the culture dish to be oocyte culture solution containing Phytohemagglutinin (PHA) of 0.3mg/mL, sparsely arranging the oocyte culture solution and waiting for adhesion;
transferring 25 enucleated oocytes to the culture dish of the first step each time, and stirring the enucleated oocytes by using a pointed glass tube to be adhered with the donor cells obtained in the first step to form a half egg-donor cell pairing body;
2) binding another enucleated clean oocyte to the donor-recipient cell counterpart:
thirdly, the other enucleated oocyte is stirred by a pointed glass tube along the gap of the half egg-donor cell pairing body (the bonding position of the enucleated oocyte and the donor cell) obtained in the second step and is bonded with the half egg-donor cell pairing body to form a plane triangle-shaped oocyte-somatic cell-oocyte pairing body (egg-donor-egg for short) with the donor cell in the middle and the enucleated oocyte at two sides;
transferring the oocyte-somatic cell-oocyte coupled body obtained in the step (III) into oocyte culture solution, bonding 15-25 oocyte-somatic cell-oocyte coupled bodies each time, transferring into electric fusion solution to be fused, so that donor cells and acceptor cells are completely fused into a whole, and the subsequent starting of a nuclear mass reprogramming program is facilitated;
the oocyte culture solution of the step 1) and the step 2) is the same as that of the example 1;
the mixture of step 2) and the electric fusion solution is added to the same manner as in example 1.
Example 3:
a method for improving the means of donor-recipient cell adhesion in a manual cloning procedure, comprising the steps of:
1) adhering enucleated clean oocytes to donor cells (porcine fetal fibroblasts):
digesting donor cells by pancreatin, wherein the pancreatin concentration is 0.25%, digesting for 5min, stopping immediately after the donor cells are rounded, digesting 50 donor cells each time, placing the donor cells in a 60mm culture dish (the culture dish is used for replacing the donor cells), and enabling the liquid in the culture dish to be oocyte culture solution containing Phytohemagglutinin (PHA) of 0.5mg/mL, sparsely arranging the oocyte culture solution and waiting for adhesion;
transferring 25 enucleated oocytes to the culture dish of the first step each time, and stirring the enucleated oocytes by using a pointed glass tube to be adhered with the donor cells obtained in the first step to form a half egg-donor cell pairing body;
2) binding another enucleated clean oocyte to the donor-recipient cell counterpart:
thirdly, the other enucleated oocyte is stirred by a pointed glass tube along the gap of the half egg-donor cell pairing body (the bonding position of the enucleated oocyte and the donor cell) obtained in the second step and is bonded with the half egg-donor cell pairing body to form a plane triangle-shaped oocyte-somatic cell-oocyte pairing body (egg-donor-egg for short) with the donor cell in the middle and the enucleated oocyte at two sides;
transferring the oocyte-somatic cell-oocyte coupled body obtained in the step (III) into oocyte culture solution, bonding 15-25 oocyte-somatic cell-oocyte coupled bodies each time, transferring into electric fusion solution to be fused, so that donor cells and acceptor cells are completely fused into a whole, and the subsequent starting of a nuclear mass reprogramming program is facilitated;
the oocyte culture solution of the step 1) and the step 2) is the same as that of the example 1;
the mixture of step 2) and the electric fusion solution is added to the same manner as in example 1.
Comparative example 1:
compared with example 1, the liquid in the culture dish of step 1) is oocyte culture liquid without Phytohemagglutinin (PHA) (i.e. 0mg/mL phytohemagglutinin PHA), compared with example 1, step 2) the enucleated and cleaned oocytes are put into operation liquid drops to stand and then are transferred into operation liquid drops containing donor cells, one oocyte is firstly adhered to one donor cell and then adhered to another oocyte, and the sequence is that the oocytes and the oocyte integrated cells (short for: egg-donor), otherwise as in example 1.
Comparative example 2:
the fluid in the culture dish of step 1) was a culture solution of oocytes of Phytohemagglutinin (PHA) at 0.4mg/mL, as compared to example 1. Compared with example 1, step 2) placing enucleated oocytes into working drops, standing, then transferring into working drops containing donor cells, first bonding one oocyte with one donor cell using 0.4mg/mL phytohemagglutinin PHA, and then bonding another oocyte together, wherein the sequence of the oocyte-oocyte integrated cells (abbreviation: egg-donor), otherwise as in example 1.
Comparative example 3:
the procedure of example 1 was otherwise the same as that of example 1 except that the culture dish in step 1) contained a culture solution of oocytes containing Phytohemagglutinin (PHA) in an amount of 0.2 mg/mL.
Comparative example 4:
the procedure of example 1 was otherwise the same as that of example 1 except that the culture dish in step 1) contained a culture solution of oocytes containing Phytohemagglutinin (PHA) in an amount of 0.6 mg/mL.
The experimental results are as follows:
comparative example and comparative example the effect on donor-receptor cell fusion efficiency of both adhesion modes:
TABLE 1 Effect of different adhesion methods on fusion efficiency
TABLE 2 efficiency of different bonding methods
And (4) analyzing results:
1. compared with comparative example 1, examples 1-3 adopt an egg-donor-egg adhesion mode, comparative example 1 adopts an egg-donor adhesion mode, examples 1-3 add Phytohemagglutinin (PHA) oocyte culture fluid to perform fusion in an egg-donor-egg adhesion mode, the fusion efficiency is as high as 88.03% + -0.58, which is remarkably higher than the fusion rate 0 of comparative example 1, and the improvement method of the donor-acceptor cell adhesion mode in the manual cloning procedure of examples 1-3 of the invention indicates that PHA is necessary in the donor-acceptor cell adhesion process.
2. Comparing examples 1-3 using egg-donor-egg binding and comparative examples 1-2 using egg-donor binding, examples 1-3 using the improved method for donor-recipient cell binding in the manual cloning procedure of examples 1-3 of the present invention and comparative examples 1-2 using the improved method for donor-recipient cell binding, the donor-recipient cell binding plays a key role in the fusion of enucleated egg cytoplasm and donor nucleus. The method for improving the donor-acceptor cell adhesion mode in the manual cloning procedure adopts the egg-donor type adhesion method in the comparative example 1-2, is simple and convenient and is beneficial to operation, but is relatively time-consuming and low in fusion efficiency; the egg-donor-egg bonding method of examples 1 to 3, which takes less time and has high fusion efficiency, requires only 23 seconds for the egg-donor-egg bonding method compared to 30 seconds for bonding a coupled body.
3. Compared with the method of comparative examples 3-4, the method of improving the donor-recipient cell adhesion pattern in the manual cloning procedure of examples 1-3 of the present invention has the advantages that the oocyte culture fluid containing Phytohemagglutinin (PHA) with different concentrations can affect the fusion rate of the egg-donor-egg adhesion pattern, the fusion efficiency of examples 1-3 is as high as 86.24-88.03%, which is significantly higher than the fusion rate of 81.77-82.69% of the oocyte culture fluid containing Phytohemagglutinin (PHA) with other concentrations of comparative examples 3-4. The reason is that the appropriate concentration of PHA (0.4mg/mL) firmly sandwiches the donor cell between the two enucleated cytoplasts, increasing the cell membrane adhesion between the donor cell and the cytoplast and between the cytoplasts, thus making the structure of the whole cell string more stable and also increasing the fusion rate due to their close contact. However, in the group to which no PHA was added during the donor-acceptor binding (comparative example 1), the fusion efficiency was 0, indicating that PHA was essential for the donor-acceptor binding, and the optimum concentration for its addition was 0.4 mg/mL.
The results show that:
the fusion rate of the egg-donor-egg type in example 1 is significantly higher than that of the egg-donor-egg type in comparative example 1, 88.03% vs 79.95%, so the method for improving the adhesion mode of donor-acceptor cells in the manual cloning procedure of the invention is feasible, and is worthy of popularization and application.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Claims (8)
1. Method for improving the means for adhesion of donor-recipient cells in a manual cloning procedure, characterized in that: the method comprises the following steps:
1) adhering enucleated clean oocytes to donor cells:
digesting donor cells by pancreatin, immediately stopping the round turning of the donor cells, digesting 50-60 donor cells each time, putting the donor cells into a culture dish after the donor cells are round, wherein the liquid in the culture dish is oocyte culture solution containing 0.3-0.5mg/mL phytohemagglutinin, and sparsely arranging the donor cells to wait for adhesion;
transferring the enucleated and clean oocyte 20-40 times to the culture dish of the first step each time, and stirring the enucleated and clean oocyte by using a pointed glass tube to be adhered with the donor cell obtained in the first step to form a half egg-donor cell pairing body;
2) binding another enucleated clean oocyte to the donor-recipient cell counterpart:
shifting the other denucleated oocyte along the gap of the half egg-donor cell paired body obtained in the step two by using a pointed glass tube, and adhering the denucleated oocyte and the half egg-donor cell paired body to form a flat triangular oocyte-somatic cell-oocyte paired body with the donor cell in the middle and the denucleated oocyte on two sides;
transferring the oocyte-somatic cell-oocyte coupled body obtained in the step (III) into oocyte culture solution, bonding 15-25 oocyte-somatic cell-oocyte coupled bodies each time, transferring into electric fusion solution to be fused, completely fusing donor cells and acceptor cells into a whole, and facilitating the starting of the subsequent nuclear mass reprogramming program.
2. The method for improving the means for adhesion of donor-recipient cells in a manual cloning procedure according to claim 1, wherein: the donor cell in the step 1) is a porcine fetal fibroblast.
3. The method for improving the means for adhesion of donor-recipient cells in a manual cloning procedure according to claim 1, wherein: digesting with pancreatin in the step 1), wherein the concentration of the pancreatin is 0.25 percent, and the digestion time is 3-5 min.
4. The method for improving the means for adhesion of donor-recipient cells in a manual cloning procedure according to claim 1, wherein: the culture dish in the step 1) is selected from a 60mm culture dish.
5. The method for improving the means for adhesion of donor-recipient cells in a manual cloning procedure according to claim 1, wherein: the oocyte culture solution of the step 1) and the step 2) comprises 80% by volume of TCM199, 10% by volume of pig follicular fluid, 10% by volume of fetal bovine serum, 0.1g/L cysteine, 0.075g/L penicillin, 0.05g/L streptomycin, 10ng/mL insulin-like growth factor, 50ng/mL epidermal growth factor, 2.2g/LNaHCO310IU/mL pregnant mare serum gonadotropin, 10IU/mL human chorionic gonadotropin and 10-30 mu mol/L butylbenzoic acid; the main components of the electric activation liquid are: 0.3mmol/L mannitol, 0.1mmol/L CaCl2·H2O、0.1mmol/L MgSO4·7H2O, 0.5mmol/L Hepes, 0.01% w/v polyvinyl alcohol.
6. The method for improving the means for adhesion of donor-recipient cells in a manual cloning procedure according to claim 1, wherein: the oocyte culture solution of the step 1) contains phytohemagglutinin of 0.4 mg/mL.
7. The method for improving the means for adhesion of donor-recipient cells in a manual cloning procedure according to claim 1, wherein: the gap of the half egg-donor cell pairing body in the step 2) refers to the bonding position of the enucleated oocyte and the donor cell.
8. The method for improving the means for adhesion of donor-recipient cells in a manual cloning procedure according to claim 1, wherein: moving the bonded oocyte-somatic cell-oocyte paired body into a fusion groove for fusion-electric activation, applying an alternating current electric field and an alternating current electric pulse of 1-3V/mm, enabling the oocyte-somatic cell-oocyte paired body to automatically lean against an electrode, wherein an enucleated oocyte of one pole is tightly attached to one end of the electrode, an oocyte of the other pole is suspended in the fusion groove, a somatic cell is clamped between two enucleated oocytes, the oocyte-somatic cell-oocyte paired body is perpendicular to a magnetic field of the electrode, and an electric fusion parameter AC: 6V/cm; DC: 1.2kV/cm, 30 mu s, 2DC, after electrically activated reconstructed embryo is transferred into embryo culture solution and washed for 3 times, the reconstructed embryo is recovered in an incubator at 38.5 ℃ for 30min, and then the fusion condition is checked.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113174366A (en) * | 2021-03-29 | 2021-07-27 | 广西壮族自治区畜牧研究所 | In-vitro maturation system of porcine oocyte containing butylbenzohydroxy acid and application thereof |
| CN113355363A (en) * | 2021-07-02 | 2021-09-07 | 湖北省农业科学院畜牧兽医研究所 | Method for cloning embryos by batch fusion of donor cells with magnetism |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040077077A1 (en) * | 2002-10-18 | 2004-04-22 | Xiangzhong Yang | Novel methods for the production of cloned mammals, mammals cloned according to the methods, and methods of use of same |
| CN101906412A (en) * | 2010-06-12 | 2010-12-08 | 中国科学院西北高原生物研究所 | Chemical auxiliary fusion method of Tibetan antelope fibrocyte and receptor ovarian ovum |
| CN102703511A (en) * | 2012-06-10 | 2012-10-03 | 广东温氏食品集团有限公司 | Fuse method for animal body nucleus transfer |
| CN102766655A (en) * | 2012-06-20 | 2012-11-07 | 中国农业科学院北京畜牧兽医研究所 | Method for producing somatic cell cloned bovine blastocyst |
| CN113174366A (en) * | 2021-03-29 | 2021-07-27 | 广西壮族自治区畜牧研究所 | In-vitro maturation system of porcine oocyte containing butylbenzohydroxy acid and application thereof |
-
2021
- 2021-04-21 CN CN202110427855.2A patent/CN113174410A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040077077A1 (en) * | 2002-10-18 | 2004-04-22 | Xiangzhong Yang | Novel methods for the production of cloned mammals, mammals cloned according to the methods, and methods of use of same |
| CN101906412A (en) * | 2010-06-12 | 2010-12-08 | 中国科学院西北高原生物研究所 | Chemical auxiliary fusion method of Tibetan antelope fibrocyte and receptor ovarian ovum |
| CN102703511A (en) * | 2012-06-10 | 2012-10-03 | 广东温氏食品集团有限公司 | Fuse method for animal body nucleus transfer |
| CN102766655A (en) * | 2012-06-20 | 2012-11-07 | 中国农业科学院北京畜牧兽医研究所 | Method for producing somatic cell cloned bovine blastocyst |
| CN113174366A (en) * | 2021-03-29 | 2021-07-27 | 广西壮族自治区畜牧研究所 | In-vitro maturation system of porcine oocyte containing butylbenzohydroxy acid and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 吕玲燕等: "不同电融合参数对猪手工克隆重构胚胎发育效果的影响", 《湖北农业科学》 * |
| 张诺等: "影响奶牛体细胞手工克隆技术因素的研究", 《畜牧兽医学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113174366A (en) * | 2021-03-29 | 2021-07-27 | 广西壮族自治区畜牧研究所 | In-vitro maturation system of porcine oocyte containing butylbenzohydroxy acid and application thereof |
| CN113174366B (en) * | 2021-03-29 | 2023-04-11 | 广西壮族自治区畜牧研究所 | In-vitro maturation system of porcine oocyte containing butylbenzohydroxy acid and application thereof |
| CN113355363A (en) * | 2021-07-02 | 2021-09-07 | 湖北省农业科学院畜牧兽医研究所 | Method for cloning embryos by batch fusion of donor cells with magnetism |
| CN113355363B (en) * | 2021-07-02 | 2022-05-31 | 湖北省农业科学院畜牧兽医研究所 | Method for cloning embryos by batch fusion of donor cells with magnetism |
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