CN113061572A - Micro-hole improvement method for culturing embryo without zona pellucida in manual cloning procedure - Google Patents
Micro-hole improvement method for culturing embryo without zona pellucida in manual cloning procedure Download PDFInfo
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Abstract
The invention discloses a micro-cavity improvement method for culturing embryos without zona pellucida in a manual cloning procedure, which adopts a novel reagent of Phytohemagglutinin (PHA), adds low-concentration PHA in micro-cavities, enables the micro-cavities to be soaked in the PHA, enables the inner walls of the micro-cavities to be fully contacted with the PHA, manually clones embryos without zona pellucida to reconstruct under the action of the PHA, starts from the step of cleavage, ensures that blastomeres are developed by being attached to the inner walls of the micro-cavities, and obviously improves the blastocyst rate in the cavities.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of nontransparent-free nuclear transfer, in particular to a micro-hole improvement method for nontransparent-zone-free embryo culture in a manual cloning procedure.
[ background of the invention ]
In recent years, the somatic cell nuclear transfer technology of pigs has been widely applied in the biomedical field, for example: developing animal model of human gene disease and producing gene knockout pig for human xenogeneic organ transplantation. These are based on the physiological and morphological close proximity of pigs to human organs, and have high litter size and high fertility, and can provide donor cells for xenotransplantation. Since the biological and genetic characteristics of swine have a high homology with those of human, and are considered as the first choice animals for human xenogeneic organ sources, swine is an important model animal in biomedical research. The effective combination of in vitro culture technology and the gene editing technology which is developed rapidly at present not only promotes the development and application of embryo engineering, but also drives the development of the medical field.
The zona pellucida is a glycoprotein matrix surrounding the periphery of oocytes, and plays an important role in the maturation of oocytes and the development of embryos. Since the advent of manual cloning technology, one conventional concept has been subverted: zona pellucida is not necessary for the development of the embryo. The Vajta et al (2003) study showed that digestion of zona pellucida with streptokinase in the presence of serum is an efficient and non-destructive method, even though the number of oocytes is high. Many researchers have digested zona pellucida of oocytes with 3.3mg/mL streptokinase protease (Vajta et al, 2004; Li et al, 2006; Du et al, 2007).
The traditional nuclear transfer technology is carried out under the condition of a zona pellucida, expensive micromanipulation equipment is needed, the skill level of technicians is required to be relatively high, and the number of cloned embryos produced per hour by all people is limited, so that the development prospect of the technology is limited to a certain extent. In contrast, the non-transparent nuclear transfer technique has the advantages of simple operation, high production efficiency, and the like, and can produce cloned embryos with higher efficiency (Vajta et al, 2005). Vajta et al (2000) showed that the micro-point system (WOW) can effectively improve the development of embryos coated with and without zona pellucida.
The method for culturing the embryo without the zona pellucida comprises the following steps: the microdroplet method and the microvia method. The study of Vajta et al (2000) shows that the micro-hole culture method is suitable for culturing embryos with complete zona pellucida and no transparence, and the cleavage rate and blastocyst rate of the micro-hole method are both significantly higher than those of the micro-droplet method (20.98% vs 12.77%, p < 0.05). The microdroplet method of Vajta et al (2000) on cattle can culture single blastocysts without embryos with the embryo being carried by 29 percent. At present, the report of the optimal microhole size for culturing embryos without zona pellucida is rare in China, and researches of Feltrin and the like (2005) show that the cleavage rate (80.9 percent vs 64.1 percent) and the blastocyst rate (25.3 percent vs 11.9 percent) of the embryo development in the small-size microholes (the inner diameter is 130 mu m and the depth is 150 mu m) are higher than the development rate of the large-size microholes (the inner diameter is 280 mu m and the depth is 250 mu m). Research also shows that the micro-holes with smaller volume can better accommodate the blastocysts without the zona pellucida, the blastocyst rate of the blastocysts is slightly higher than that of the blastocysts with larger micro-holes, but the cleavage rate difference of the two is not obvious.
Before the embryo without the zona pellucida is reconstructed, the bottom of a four-hole plate which is added with an embryo culture solution and covered with paraffin oil is uniformly punched with an embryo gathering needle, so that each embryo is placed in 1 micro-hole, the size and the depth of the micro-hole directly influence the accommodating capacity of the embryo without the zona pellucida, and the accommodating capacity determines the development efficiency of the embryo hair. There are many procedures that affect the efficiency of manual cloning, such as enucleation of oocytes, activation of fusion of donor/recipient cytoplasm, and culture systems without zona pellucida embryos. The embryo culture system is an important link, and the development of the manually cloned embryos without the coating of the zona pellucida can not be subjected to the culture of micro-holes.
Therefore, it is very important to study the improved method of micro-cavity without zona pellucida embryo culture in the manual cloning procedure to improve the efficiency of manual cloning.
[ summary of the invention ]
Aiming at the problem of low manual cloning efficiency in the prior art, the invention provides a micro-hole improvement method for culturing embryos without zona pellucida in a manual cloning procedure.
The culture of the embryo without the zona pellucida in the manual cloning procedure comprises the following steps:
1) maturation of oocytes: washing pig ovary with 75% ethanol for 15s, then washing 3 times with physiological saline added with penicillin and streptomycin at 37 ℃, then extracting follicle with 3-8mm on the surface of the ovary with a 10mL injector, standing for 15min, discarding supernatant, selecting Cumulus Oocyte Complexes (COCs) with good refractivity and coated by 3 layers or more of particles/cumulus cells under a stereoscopic microscope, putting the cumulus oocyte complexes into a mature solution for culturing (containing 10IU/mL hCG and 10IU/mL eCG), and after culturing for 20-22h, changing the culture solution into a culture solution without hormone for further culturing for 18-20 h;
2) culturing donor cells: obtaining pig fetus from slaughterhouse, cleaning with normal saline, transferring to aseptic super clean bench, separating ear tissue, cleaning with PBS containing antibiotic for 2 times, placing into 60mm aseptic plate, cutting into 2-3mm tissue blocks, adding appropriate amount of DMEM culture solution containing 10% FBS into culture dish, spreading the tissue blocks uniformly with tweezers, marking on dish cover, and back-buckling at 37 deg.C and 5% CO2In the incubator, a small amount of culture solution is added after 4 hours to prevent the tissue block from floating. Filling culture solution at the bottom of the dish after 2h, observing the growth condition of the fibroblasts after 48h, carrying out passage when the fibroblasts grow to about 90% and are converged, transferring the fibroblasts to 5-6 generations for serving as the pig nuclear transplantation donor cells;
3) enucleating high-quality oocytes: oocytes that are uniform in cytoplasm, good in refractivity, and contain the first polar body were placed in 1% hyaluronidase, and granulosa cells were removed. Then transferring the oocyte into a pronase solution of 3.3mg/mL, when the zona pellucida of the oocyte is observed to start to deform, immediately transferring the oocyte into a washing solution (T20) containing 20% fetal calf serum to wash for 3 times, and when the oocyte is recovered to be circular, transferring the oocyte with complete recovery and better quality into a cutting solution containing 2.5 mu g/mL Cytochalasin B (CB) to denucleate by using a hard glass pipette with the caliber of 200 and 300 mu M;
chemical-assisted enucleation: incubating the selected high-quality oocyte in 0.4 μ g/mLDC (decarbonylation colchicine) for 45min, then digesting a part of zona pellucida, after the zona pellucida is recovered to be circular, placing the protrusion or polar body at the position of 6 points or 12 points, removing the cytoplasm containing the protrusion or polar body about 1/3 by using a cutting knife, and leaving 2/3 cytoplasm as a receptor cytoplasm;
4) fusion activation of donor/receptor cytoplasm:
a: bonding: the specific method comprises the following steps: egg-donor-egg bonding
In a first step, enucleated hemi-eggs are bound to donor cells in an enucleated liquid containing Phytohemagglutinin (PHA) at 0.4mg/mL to form a hemi-egg-donor cell pair; secondly, adhering another enucleated half egg with a counterpart to form a half egg-somatic cell-half egg counterpart, and transferring the half egg-somatic cell-half egg counterpart into T20, wherein 20 counterparts are adhered each time;
b: fusion/electrical activation: moving the bonded pair into an electrofusion tank, applying an Alternating Current (AC) pulse of 1-3V/mm to make the pair automatically lean against an electrode, so that the pair is perpendicular to a magnetic field, and the electrofusion parameters are AC: 6V/cm; DC (direct current): 1.2kV/cm, 30. mu.s, 2 DC. Moving the electrically activated reconstructed embryo into T20 for washing for 3 times, recovering in an incubator for 30min, and then checking the fusion condition;
c: chemical activation: after completion of fusion, the reconstituted embryos were washed 3 times in activating solution (PAF) containing 5. mu.g/mL CB and 10. mu.g/mLCHX (cycloheximide). Then 1 reconstructed embryo is put into each drop of the activation solution and activated for 4 hours;
5) culturing reconstructed embryo without zona pellucida: the activated reconstituted embryos are washed 3 times in PZM-3 (embryo culture) before being transferred to PZM-3 containing microwells equilibrated in an incubator.
The invention relates to a micro-hole improvement method for culturing embryos without zona pellucida in a manual cloning procedure, which comprises the following steps:
1) adding embryo culture solution into four-hole plate before pricking, and pricking into 3 groups of micro-holes with the following parameters:
group 1 parameters: the diameter of the bottom is 120 +/-10 mu m, the diameter of the upper opening is about 150 +/-10 mu m, and the depth is 1500 +/-10 mu m (group 1 for short);
group 2 parameters: the diameter of the bottom is 120 +/-10 mu m, the diameter of the upper opening is about 200 +/-10 mu m, and the depth is 200 +/-10 mu m (group 2 for short);
group 3 parameters: the diameter of the bottom is 150 +/-10 mu m, the diameter of the upper opening is about 200 +/-10 mu m, and the depth is 200 +/-10 mu m (group 3 for short);
2) after pricking, pouring all the liquid in the four pore plates, and reversing for 4-6 minutes to ensure that all the liquid in the micro-holes is poured out;
3) adding Phytohemagglutinin (PHA) 0.15-0.25mg/mL into the micro-cavity to ensure that the micro-cavity is filled, soaking for 8-12 min, then slowly adding embryo culture solution into the position without the micro-cavity in the four-hole plate, and finally covering a layer of paraffin oil.
In the invention:
the step 1) of binding 3 groups of micro-holes with the following parameters according to the requirements is as follows:
group 1 parameters: the diameter of the bottom is 120 μm, the diameter of the upper opening is about 150 μm, and the depth is 150 μm;
group 2 parameters: the diameter of the bottom is 120 μm, the diameter of the upper opening is about 200 μm, and the depth is 200 μm;
group 3 parameters: the diameter of the bottom is 150 μm, the diameter of the upper opening is about 200 μm, and the depth is 200 μm;
the embryo culture solution in the step 1) comprises the following components in concentration: 6.31g/LNaCl, 0.74g/LKCl, 0.047g/LKH2PO4、2.1g/LNaHCO30.022g/L of sodium pyruvate, 0.62g/L of calcium lactate, 0.15g/L L-glutamine, 0.55g/L of hypotaurine, 20ml/L of essential amino acid, 10ml/L of nonessential amino acid, 0.065g/L of penicillin, 0.05g/L of streptomycin and 4g/L of fetal bovine serum albumin.
And (3) reversing the process in the step 2), wherein the time is 5 minutes.
And 3) soaking for 10 minutes.
Compared with the prior art, the invention has the following advantages:
1. the invention relates to a micro-hole improvement method for culturing embryos without zona pellucida in a manual cloning procedure, which comprises the following steps: adding low-concentration 0.2mg/mL PHA into the micro-cavity, soaking the micro-cavity in the PHA, fully contacting the inner wall of the micro-cavity with the PHA, manually cloning a nontransparent zone-free reconstructed embryo under the action of the PHA, starting from the cleavage step, attaching a blastomere to the inner wall of the micro-cavity to develop, so that the cleavage rate in the cavity and the blastocyst rate in the cavity are both obviously higher than those in a comparative example.
2. The invention explores the optimal PHA concentration (0.2mg/mL) for improving the development rate of the manual cloning of the zona pellucida-free embryo in the cave; the size of a specific micro-cavity (bottom diameter 120 μm, top diameter 200 μm, depth 200 μm) suitable for manual cloning of zona pellucida-free embryo culture was explored.
[ description of the drawings ]
FIG. 1 is a diagram of micro-holes punched out in example 1 of the present invention.
FIG. 2 is a drawing of a needle for collecting embryos inserted into micro-cavities according to 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:
the improved method of micro-hole without zona pellucida embryo culture in manual cloning procedure includes the following steps:
1) adding embryo culture solution into four-hole plate before pricking, and pricking into 3 groups of micro-holes with the following parameters:
group 1 parameters: the diameter of the bottom is 120 μm, the diameter of the upper opening is about 150 μm, and the depth is 150 μm (group 1 for short);
group 2 parameters: the diameter of the bottom is 120 μm, the diameter of the upper opening is about 200 μm, and the depth is 200 μm (group 2 for short);
group 3 parameters: the diameter of the bottom is 150 μm, the diameter of the upper opening is about 200 μm, and the depth is 200 μm (group 3 for short);
2) after pricking, pouring all the liquid in the four pore plates, and reversing for 5 minutes to ensure that all the liquid in the micro-holes is poured out;
3) adding Phytohemagglutinin (PHA) 0.2mg/mL into the micro-wells to ensure that the micro-wells are filled, soaking for 10 min, then slowly adding embryo culture solution into the positions without micro-wells in the four-well plate, and finally covering with a layer of paraffin oil (see figure 1).
The results are shown in Table 1.
Example 2:
the improved method of micro-hole without zona pellucida embryo culture in manual cloning procedure includes the following steps:
1) adding embryo culture solution into four-hole plate before pricking, and pricking into 3 groups of micro-holes with the following parameters:
group 1 parameters: the diameter of the bottom is 130 μm, the diameter of the upper opening is about 140 μm, and the depth is 150 μm;
group 2 parameters: the diameter of the bottom is 130 μm, the diameter of the upper opening is about 190 μm, and the depth is 210 μm;
group 3 parameters: the diameter of the bottom is 160 μm, the diameter of the upper opening is about 190 μm, and the depth is 210 μm;
2) after pricking, pouring all the liquid in the four pore plates, and reversing for 4 minutes to ensure that all the liquid in the micro-holes is poured out;
3) adding Phytohemagglutinin (PHA) 0.15mg/mL into the micro-wells to ensure that the micro-wells are filled, soaking for 8 minutes, then slowly adding embryo culture solution into the positions without the micro-wells in the four-well plate, and finally covering with a layer of paraffin oil.
Example 3:
the improved method of micro-hole without zona pellucida embryo culture in manual cloning procedure includes the following steps:
1) adding embryo culture solution into four-hole plate before pricking, and pricking into 3 groups of micro-holes with the following parameters:
group 1 parameters: the diameter of the bottom is 110 μm, the diameter of the upper opening is about 160 μm, and the depth is 140 μm;
group 2 parameters: the diameter of the bottom is 110 μm, the diameter of the upper opening is about 210 μm, and the depth is 190 μm;
group 3 parameters: the diameter of the bottom is 140 μm, the diameter of the upper opening is about 210 μm, and the depth is 190 μm;
2) after pricking, pouring all the liquid in the four pore plates, and reversing for 6 minutes to ensure that all the liquid in the micro-holes is poured out;
3) adding Phytohemagglutinin (PHA) 0.25mg/mL into the micro-cavity to ensure that the micro-cavity is filled, soaking for 12 min, then slowly adding embryo culture solution into the position without the micro-cavity in the four-hole plate, and finally covering a layer of paraffin oil.
Comparative example 1:
compared with example 1, step 3) was not added with Phytohemagglutinin (PHA) as a control group, and the other steps were the same as example 1.
Comparative example 2:
step 3) was performed by adding Phytohemagglutinin (PHA) in an amount of 0.3mg/mL as compared with that in example 1, and the procedure was otherwise the same as in example 1.
Comparative example 3:
step 3) was performed by adding 0.3mg/mL of polyvinyl alcohol (PVA) as compared with example 1, and the procedure was otherwise the same as in example 1.
The results are shown in Table 1:
note: the data in the same column are marked with different letters to represent that the data in the same column are different remarkably (P <0.05), and no letters or the same letters are included to represent that the data are not different remarkably (P > 0.05).
As can be seen from Table 1:
1) the effect of PHA addition on development of manually cloned zona pellucida embryos in different micro-cavity parameters was studied in comparison of example 1 and comparative example 1. example 1 shows that culture groups containing PHA liquid in micro-cavities have significantly higher hole cleavage rates (87.04%, 88.27%, 85.69% VS 82.52%) and higher hole blastocyst rates (29.19%, 30.37%, 27.24% VS 20.68%) than comparative example 1 (control group) under the micro-cavity parameters of groups 1, 2 and 3 of example 1. For blastocyst cell counts, the micro-cavities contained no PHA fluid, which was not very different.
2) The effect of different PHA addition amounts on the development effect of manually cloned zona pellucida embryos in different micro-hole parameters is researched by comparing example 1 with comparative example 2, and the comparative example 2 shows that the 0.2mg/mL PHA can more remarkably improve the in-hole cleavage rate and the in-hole blastocyst rate of manually cloned zona pellucida embryos compared with 0.3mg/mL PHA.
3) The effect of adding different reagents into the micro-cavities on the development effect of manually cloned zona pellucida embryos in different micro-cavity parameters is researched by comparing example 1 with comparative example 3, and the comparative example 3 shows that the in-cavity cleavage rate and the in-cavity blastocyst rate of manually cloned zona pellucida embryos can be improved more remarkably by 0.2mg/mL PHA compared with 0.3mg/mL PVA.
Reason analysis:
1) the micro-cavity of example 1 was soaked in PHA with a low concentration of 0.2mg/mL PHA, the inner wall of which was in full contact with PHA, and the embryos were reconstituted by manual cloning of zona pellucida under the action of PHA, and from the stage of cleavage, blastomeres developed against the inner wall of the micro-cavity, so that the cleavage rate at the cavity, and the rate of blastocysts at the cavity were significantly higher than those of the comparative example (control).
2) The micro-holes with smaller volume can better accommodate the manually cloned embryos without zona pellucida compared with the micro-holes with larger volume, and the analysis is that the size of the micro-holes is identical with the size of the blastula, so the embryos can well develop in the micro-holes.
3) The accommodation capacity of PHA and PVA in other concentrations for manually cloning the zona pellucida embryos in the hole is not as strong as that of PHA of 0.2mg/mL, so that PHA of 0.2mg/mL can improve the cleavage rate and blastocyst rate of the zona pellucida embryos manually cloned in the hole.
And (4) conclusion:
1) PHA with low concentration is added into micro-holes, so that the in-hole cleavage rate and in-hole blastocyst rate of manually cloned embryos without zona pellucida can be improved;
2) under the group 2 micro-hole parameters (the bottom diameter is 120 μm, the upper opening diameter is about 200 μm, and the depth is 200 μm), the manually cloned zona pellucida-free embryo has the highest hole cleavage rate and hole blastocyst rate among the three groups of micro-hole parameters, and is the micro-hole parameter which is worth popularizing and applying.
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 (5)
1. The improved method of micro-hole without zona pellucida embryo culture in manual cloning procedure is characterized by comprising the following steps: the method comprises the following steps:
1) adding embryo culture solution into four-hole plate before pricking, and pricking into 3 groups of micro-holes with the following parameters:
group 1 parameters: the diameter of the bottom is 120 +/-10 mu m, the diameter of the upper opening is about 150 +/-10 mu m, and the depth is 1500 +/-10 mu m;
group 2 parameters: the diameter of the bottom is 120 +/-10 mu m, the diameter of the upper opening is about 200 +/-10 mu m, and the depth is 200 +/-10 mu m;
group 3 parameters: the diameter of the bottom is 150 +/-10 mu m, the diameter of the upper opening is about 200 +/-10 mu m, and the depth is 200 +/-10 mu m;
2) after pricking, pouring all the liquid in the four pore plates, and reversing for 4-6 minutes to ensure that all the liquid in the micro-holes is poured out;
3) adding phytohemagglutinin 0.15-0.25mg/mL into the micro-holes to ensure that the micro-holes are filled, soaking for 8-12 min, slowly adding embryo culture solution into the positions without the micro-holes in the four-hole plate, and covering with a layer of paraffin oil.
2. The method of improving micropockets for zona pellucida-free embryo culture in a manual cloning procedure of claim 1, wherein: the step 1) of binding 3 groups of micro-holes with the following parameters according to the requirements is as follows:
group 1 parameters: the diameter of the bottom is 120 μm, the diameter of the upper opening is about 150 μm, and the depth is 150 μm;
group 2 parameters: the diameter of the bottom is 120 μm, the diameter of the upper opening is about 200 μm, and the depth is 200 μm;
group 3 parameters: the diameter of the bottom is 150 μm, the diameter of the upper opening is about 200 μm, and the depth is 200 μm.
3. The method of improving micropockets for zona pellucida-free embryo culture in a manual cloning procedure of claim 1, wherein: the embryo culture solution in the step 1) comprises the following components in concentration: 6.31g/LNaCl, 0.74g/LKCl, 0.047g/LKH2PO4、2.1g/LNaHCO30.022g/L of sodium pyruvate, 0.62g/L of calcium lactate, 0.15g/L L-glutamine, 0.55g/L of hypotaurine, 20ml/L essential amino acid, 10ml/L nonessential amino acid, 0.065g/L penicillin, 0.05g/L streptomycin and 4g/L fetal bovine serum albumin.
4. The method of improving micropockets for zona pellucida-free embryo culture in a manual cloning procedure of claim 1, wherein: and (3) reversing the process in the step 2), wherein the time is 5 minutes.
5. The method of improving micropockets for zona pellucida-free embryo culture in a manual cloning procedure of claim 1, wherein: and 3) soaking for 10 minutes.
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