CN105200086A - Preparation method of selectable marker-free polygenes complex character genetic engineering pig - Google Patents

Abstract

The present invention relates to the field of transgenic pigs, in particular to a method for preparing genetically engineered pigs with multigene complex traits without selectable markers, comprising the following steps: Fibroblasts are used as donors, enucleated mature porcine oocytes are used as recipients, and somatic cell nuclear transfer is used to obtain reconstituted embryos containing multigene complex trait genes without selection markers; after in vitro culture, transplanted into surrogate In pigs, after the pigs give birth, the genetically engineered pigs with multiple genes and complex traits without selection markers can be obtained. The method is to prepare sFat1-2A-Fad2 transgenic pigs by somatic cell nuclear transfer of genes without selection markers, and obtain transgenic pigs rich in n-3 unsaturated fat, thereby speeding up the improvement of pig meat quality; and the transferred genes There is no selection marker gene, which avoids the potential harm brought by the selection marker gene.

Description

A method for preparing genetic engineering pigs with multiple genes and complex traits without selection markers

technical field

The invention relates to the field of transgenic pigs, in particular to a method for preparing genetically engineered pigs with no selection markers and multigene complex traits.

Background technique

Pig is one of the important livestock animals and an important source of meat in our country, which is closely related to people's daily life. With the rapid development of transgenic technology, pigs have been pushed to the stage of breeding improvement, medical application, and commercial operation, and they are in an unprecedented opportunity.

Although transgenic animals are one of the most promising topics, the low efficiency of making transgenic animals and the potential risks of animal welfare and food safety brought about by using antibiotic resistance genes as selection markers are common problems and are the main problems at present. restrictive factors. At present, animal genetic engineering methods only integrate one target gene for each transgene, and then prepare genetically engineered animals expressing a single gene. However, the actual situation is that the characteristics of animals are often not determined by one gene, that is, one characteristic of an animal is related to multiple genes. , or, in reality, genetically engineered animals are required to have more than two genetic characteristics, which we call composite traits. In actual production, genetically engineered animals with multiple genes and complex traits are more practical. If it involves the preparation of genetically engineered animals with polygenic complex traits, it is necessary to separately prepare corresponding single-gene genetically engineered animals, and then prepare the offspring of transgenic animals through methods such as mating among genetically engineered animals. This method of preparing polygenic animals Time consuming and costly. The selection marker gene is accompanied by the target gene, and whether the target gene is successfully transferred is determined by the expression of the selection marker gene. However, the selectable marker gene no longer plays any role after the cells have been transferred to the medium or recipient. In recent years, researchers have proposed that the existence of selectable marker genes has unpredictable hazards to human health and the environment, and may destroy the ecological balance of populations and affect genetic diversity. For transgenic animals, selectable marker genes mainly include antibiotic resistance genes, such as kanamycin resistance gene, neomycin resistance gene and streptomycin resistance gene for aminoglycoside antibiotic resistance. Animal food derived from genetically modified animals may allow antibiotic resistance gene products to enter the human body through the food chain, and inactivate ordinary doses of antibiotics in the ecological environment, posing a threat to human public health and even the entire ecological environment.

Considering the drawbacks of selectable marker genes, the best way to overcome this problem is to remove the selectable marker gene. At present, there are many studies on plants. For example, the Agrobacterium-mediated co-transformation method inserts the target gene and the selectable marker gene into different T-DNAs, and jointly transfers and screens out transgenic plants. Plants with selectable marker genes.

Site-specific recombination is used to delete selectable marker genes under inducible promoters. The most widely used system is the Cre/loxP system. The cre gene product of Escherichia coli P1 phage can recognize the loxP site and catalyze the recombination of the two. When the two are specifically recognized and combined, the expression of the target gene can be initiated. After the transgenic crops or animals are screened out, offspring with selectable marker genes need to be eliminated through hybridization; antibiotic resistance genes can also be eliminated at the cellular level by identifying loxP sites.

The safe selectable marker gene refers to the use of selectable marker genes other than non-antibiotic resistance genes. The selection of this marker gene is based on the fact that the protein encoded by it is not metabolized by the receptor, and the transformed cell has favorable conditions for metabolism and development. For example, plants convert hexose mannose into mannose-6-phosphate. Due to the lack of related enzymes for metabolizing mannose-6-phosphate, mannose-6-phosphate cannot be further utilized, and PMI catalyzes mannose-6 - Phosphoric acid is converted to fructose, the precursor of the glycolytic pathway. Weisser et al. (1996) used mannose phosphate isomerase PMI as a selectable marker gene to give transformed cells a metabolic advantage, while non-transgenic cells were still unable to metabolize mannose-6-phosphate. Similar safe selection marker genes include xylose isomerase xylA and ribitol operon rtl. In addition to sugar metabolism genes, there are hormone synthesis-related genes and GFP genes based on visual selection. Most of these marker genes are derived from animals and plants, and their mechanism of action is similar to that of traditional marker genes, but they do not cause potential harm to organisms and the environment. Their use methods are relatively mature, and some marker genes can also be expressed as target genes.

In view of this, the present invention is proposed.

Contents of the invention

The object of the present invention is to provide a method for preparing genetically engineered pigs with multigene complex traits without selectable markers, to create genetically edited animal pigs with multigene complex traits without selectable markers, and to realize the ability of Δ12 fatty acid desaturase (Fad-2 ) and the nematode (C.briggsae) ω-3 fatty acid desaturase gene (sFat-1) were simultaneously transferred. On the basis of obtaining positive cells transfected with sFat1-2A-Fad2 without selection markers, adopt the method of somatic cell nuclear transfer to prepare sFat1-2A-Fad2 transgenic pigs, and obtain transgenic pigs rich in n-3 unsaturated fat, thereby speeding up the development of pigs. It is of great practical significance to improve the speed of meat quality improvement and provide human beings with more nutritious pork.

In order to realize the above-mentioned purpose of the present invention, special adopt following technical scheme:

A method for preparing a genetically engineered pig with multiple genes and complex traits without a selection marker, comprising the following steps:

Using fibroblasts carrying sFat-1-2A-Fad-2 gene fragments without selectable markers as donors and enucleated mature porcine oocytes as recipients, the somatic cell nuclear transfer method was used to obtain cells containing non-selectable markers Reconstructed embryos of polygenic compound trait genes;

After the reconstituted embryo is cultured in vitro, it is transplanted into a surrogate pig, and after the pig is delivered, a genetically engineered pig with multiple genes and complex traits without selection markers can be obtained.

The invention provides a method for preparing genetically engineered pigs with multigene complex traits without selectable markers, which is to prepare the sFat1-2A-Fad2 transgene by adopting somatic cell nuclear transfer of the sFat-1-2A-Fad-2 gene without selectable markers Pigs, obtain transgenic pigs rich in n-3 unsaturated fat, so as to speed up the improvement of pig meat quality and provide human beings with more nutritious pork; and the transferred genes do not have selection marker genes, avoiding selection marker genes. potential hazards.

Preferably, follicles with a diameter of 5-8 mm are selected to extract oocytes, and the oocytes are matured in vitro to obtain mature porcine oocytes. The in vitro maturation rate and quality of porcine oocytes are closely related to the size of the follicle. When the diameter of the oocyte is less than 3mm, it does not have the ability to mature in vitro; when the diameter of the oocyte is greater than 6mm, aging may occur after later culture. Follicles with a diameter of 5-8mm are specifically selected to extract oocytes, and the oocytes obtained from the culture have a high maturity rate and good quality.

Preferably, the oocytes are matured in vitro to obtain mature porcine oocytes specifically:

The oocytes were cultured at 38.5±0.5° C., 5%±1% CO ₂ , and saturated humidity for 39-41 hours.

The mature oocytes cultured through this step have good vigor and are beneficial to subsequent operations.

Preferably, the oocyte used in the enucleated mature porcine oocyte is an oocyte that grows well and discharges the first polar body; Acidase solution was repeatedly pipetted to remove cumulus cells.

Preferably, the cryopreserved donor cells are recovered on the day of ovary collection;

When resuscitating cryopreserved donor cells, quickly remove the cells from liquid nitrogen and place them in a water bath at 38±1°C with constant shaking, and the cells will melt within 1 min;

Transfer the thawed cells to a centrifuge tube and add 37±1°C preheated complete medium, pipette and mix well, then centrifuge, the obtained cells are resuspended, inoculated into 37±1°C preheated complete medium for culture, culture After 24±1 hours, wash 1-2 times with DPBS, and then change the medium once;

After culturing until the cell density reaches 78%-82%, the cells are digested, and then the cell nucleus transfer operation can be performed.

Preferably, the method of somatic cell nuclear transfer is specifically:

Mature oocytes are enucleated to remove the first polar body, and then transferred to the operating drop bar;

Select recipient cells with medium size, strong refraction, round shape, dense cytoplasm, and good vitality, inhale into the injection needle, and place the injection needle in the cell operation drop;

Hold the fixed needle and place it under the enucleated oocyte, and give the force to suck the cell tightly. After the cell is sucked tightly, adjust the focal length so that the enucleated oocyte and the injection needle are on the same plane, and slightly push the injection needle outward The force of the recipient cells, when the recipient cells move to the injection needle, quickly penetrate the perivitelline space of the oocyte, and quickly remove the injection needle after the recipient cells enter the oocyte to obtain the reconstructed oocyte;

The reconstituted egg cells are activated and fused, and cultured to obtain reconstituted embryos.

Preferably, the activation of the reconstituted oocytes specifically includes: putting the reconstituted oocytes into the H199 operating solution, washing them for 1-2 times, putting them in the activation solution to balance for 1-3 minutes, and after putting them in, the reconstituted oocytes sink and take out quickly;

Put the activation solution in the fusion tank, and after warming up twice with air shock, put the reconstituted egg cells into the activation tank;

The H199 operating fluid and the activation fluid are preheated before use.

Preferably, the fusion is performed by means of electric shock;

The electric shock is performed by a fusion instrument, and the parameters of the fusion instrument are: 2.0kv/cm, 50us.

Preferably, the fusion specifically includes: adjusting the direction of the reconstructed oocyte so that the contact surface between the donor cell and the oocyte is perpendicular to the direction of the current, and shocking once;

Remove after electric shock, place in H199, wash three times, move to embryo culture solution and rinse 3-4 times, put in 38±0.5°C, 5%±1% CO ₂ incubator for cultivation.

Preferably, the surrogate pig is a healthy Landrace sow with 2-3 consecutive estrous cycles, and the estrus time is controlled within 48 hours.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art.

Fig. 1 is the schematic diagram of MLC carrier in the embodiment 1 of the present invention;

Fig. 2 is the oocyte microscope 40 × observation figure that there are more than 3 layers of cumulus cells closely surrounded by the periphery in Example 2 of the present invention;

40 × observation diagram of mature oocyte microscope in Fig. 3 of the present invention in Example 2;

Figure 4 is a diagram of the mature oocyte nuclei transfer process in Example 2 of the present invention;

Fig. 5 is a 40× observation diagram of cell division 24h morphological microscope in Example 2 of the present invention;

Fig. 6 is a 40× observation diagram of cell division 48h morphological microscope in Example 2 of the present invention;

Fig. 7 is the morphological microscope 40× observation diagram of the 6d blastocyst in Example 2 of the present invention;

Fig. 8 is positive plasmid DNA electrophoresis figure in the embodiment of the present invention 4;

Fig. 9 is the DNA electrophoresis diagram of pig ear tissue in Example 4 of the present invention;

Figure 10 is the electrophoresis diagram of PCR products of different genes in Example 4 of the present invention, A is sFat1; B is Fad2; C is 2A sequence;

11 is an agarose gel electrophoresis image of RNA extracted from different tissues of transgenic pigs in Example 4 of the present invention;

Fig. 12 is a histogram of QPCR detection results of RNA expression levels of sFat1 gene and Fad2 gene in different tissues of transgenic pigs in Example 3 of the present invention.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Example 1

Construction of sFat-1 and Fad-2 expression vectors

1. Gene synthesis

References The CDS region of the Fad-2 gene (GenBank: AB094415.1) was obtained through NCBI, as shown in SEQ ID No.1 and FMDV2A sequence in the sequence listing. After optimizing the FMDV2A sequence according to the pig's preference, the codon-optimized 2A sequence (as shown in SEQIDNo.2 in the sequence list) was obtained, and the 2A+Fad-2 sequence was artificially synthesized, and the artificially synthesized fragment was completed by Jinweizhi Company.

2. Obtaining of sFat-1 CDS fragment

sFat-1CDS (as shown in SEQIDNo.3 in the sequence listing) removes the stop codon TAA and then joins with the 2A+Fad-2 sequence, so as to prevent the translation of the stop codon of the upstream gene from causing the normal translation and expression of the downstream gene. No restriction site was added between the sequences to avoid affecting the normal translation and expression of the gene.

Obtaining of sFat-1 CDS fragment:

The pGH-sFat-1 linked with the sFat-1 CDS was double-digested with EcoRI and HindIII restriction enzyme sites. The enzyme digestion system is as follows:

pGH-sFat-12 μg, NEBBuffer 42 μl, EcoRI 0.5 μl, HindIII 0.5 μl, and ddH ₂ O to make up to 20 μl.

The enzyme digestion system was incubated overnight in a 37°C constant temperature water bath. Then electrophoresis was carried out on 1% agarose gel to obtain the target fragment, and the fragment of the target piece was recovered by using a DNA gel recovery kit. Specific steps are as follows:

a. Cut off the gel block of the target band under the gel cutter, weigh it and put it into a 1.5ml EP tube;

b. Add three times the volume of ADB to the EP tube (for example, add 300 μl ADB to 100 μl (mg) gel);

c. Place the EP tube in a dry thermostat at 37-55°C for 5-10 minutes until the gel is completely dissolved;

d. Transfer the dissolved agarose gel solution to the Zymo-Spin column, and put the column into the collection tube;

e. Centrifuge at 10000g for 30-60s, discard the liquid in the collection tube;

f. Add 200μl WashBuffer to the column, centrifuge at 10000g for 30s, discard the liquid in the collection tube; repeat this step;

g. Add >6μl ultrapure water to the column, put the column into a new 1.5ml EP tube, centrifuge at 10000g, and elute DNA for 30-60s.

The stop codon TAA of sFat-1CDS was deleted by site-directed mutagenesis, and the sFat-1CDS and 2A+Fad-2 sequence without the stop codon TAA were connected by PCR method, and then connected into the backbone vector to form MLC-sFat-1-Fad -2 carrier.

3. sFat-1-2A-Fad-2 linked backbone carrier

The MLC plasmid was digested with EcoRI and HindIII. The enzyme digestion system is as follows:

The enzyme digestion system was incubated overnight in a constant temperature water bath at 37°C, and then electrophoresed on a 1% agarose gel to obtain linearized plasmid fragments, which were recovered using a DNA gel recovery kit.

4. The company provides the sFat-1+2A+Fad-2 sequence connected to the PMD-18T vector, which is digested and recovered through the same restriction site, and then the sFat-1+2A+Fad-2 sequence is combined with the backbone vector ( As shown in Figure 1), the reaction system is as follows:

The connection system was placed in a dry thermostat at 16°C overnight.

The connection product is transformed, and the specific steps are as follows:

Add all 10 μl of the system to 100 μl competent cells (5α), mix well and incubate on ice for 30 minutes; immediately heat shock at 42°C for 90 seconds in a constant temperature water bath; immediately incubate on ice for 2 minutes; add 1000 μl of non-resistant LB liquid medium, and Place the EP tube on a constant temperature shaker at 37°C for 45-60min. Take a prepared solid culture dish added with ampicillin resistance and sterilize it under a UV lamp for 15 minutes, put a glass applicator on an alcohol lamp to sterilize, use a micropipette to take 100 μl of the mixed solution and gently blow it on the surface of the solid medium, and use Spread the glass coating rod evenly and then place it in a constant temperature incubator at 37°C for 12-16h.

The formula of 100ml LB medium is as follows:

Use the common plasmid mini-extraction kit to extract common plasmids from 1ml of bacterial liquid, and refer to the kit instructions for specific steps.

The plasmid was digested with EcoRI and HindIII, and then electrophoresed in 1% agarose gel to identify whether the ligation system constituted a double-gene expression vector, and verified by sequencing.

After the sequencing is completed, digest with Psp1406 to obtain a linearized recombinant vector for future use.

The general idea of vector construction is as follows: the downstream of the MLC1Promoter of the MLC vector contains EcoRI and HindIII restriction sites, the sFat-1 CDS is obtained by restriction digestion, the 2A+Fad-2 sequence is artificially synthesized, and the stop codon TAA of the sFat-1 CDS is deleted by site-directed mutagenesis , using the PCR method to splice the two sequences into the backbone vector to form the MLC-sFat-1-Fad-2 vector.

5. Cell culture

The primary culture of adult large white pig ear tissue fibroblasts was carried out by tissue block culture method, and the primary cells were used to prepare positive cells transfected with sFat-1 and Fad-2 genes after passage.

5.1 Solution configuration:

5.1.1 Preparation of DMEM base fluid:

a. DMEM powder: 13.4g; NaHCO ₃ powder: 3.7000g;

b. Use a volumetric flask to make up 1000ml of distilled water, then transfer the distilled water to a 1000ml beaker, and add the above powder while stirring on a magnetic stirrer until it is completely dissolved.

5.1.2 Preparation of cell culture medium:

a. Use a disposable or sterilized measuring device to measure 100ml of FBS and 10ml of double antibody;

b. Add the above liquid into a 1000ml beaker, and then add an appropriate amount of the DMEM solution prepared above;

c. Make a constant volume in a 1000ml volumetric flask;

d. Transfer the liquid in the volumetric flask to an empty 1000ml beaker and mix on a magnetic stirrer;

e. Determination of the pH of the culture medium

If the pH is alkaline, adjust the pH to 7.3 with HCl (5M);

If the pH is acidic, adjust the pH to 7.3 with NaOH (5M).

f. After adjusting the pH, measure the osmotic pressure of the cell culture medium, and the osmotic pressure suitable for the survival of the cells is 280-320;

g. Filter and aliquot to obtain 1000ml of 10% cell culture solution and store at 4°C.

5.1.3 Preparation of cell washing solution:

a. 500ml of DPBS, add 5mL double antibody;

b. Filtration and packaging.

5.1.4 Preparation of cell digestion solution:

Steps for preparing 100ml cell digestion solution:

a. Take 100ml DPBS in a 200ml beaker, add 0.02g EDTA, heat on a magnetic stirrer (60-70°C) to dissolve;

b. After cooling to room temperature, dilute to 100ml again;

c. After constant volume, place in a 200ml beaker, add 0.25g Trypsin, and stir slowly on a magnetic stirrer to dissolve it;

d. Adjust the pH to 8.0 with NaOH, and measure the osmotic pressure;

e. Filter and aliquot, store at -20°C, and thaw when used.

5.1.5 Preparation of cell cryopreservation solution:

Steps for preparing 100ml cell freezing solution:

a. Take 15ml of PBS and 10ml of DMSO in a 150ml beaker, add an appropriate amount of DMEM solution and mix well;

b. Transfer to a 100ml volumetric flask, and dilute to volume with DEME solution;

c. Filter and aliquot and store at 4°C.

5.1.6 Preparation of tissue block washing solution:

a. 1 bottle of penicillin (800,000 units/bottle) was dissolved in 4ml of normal saline;

b. 1 bottle of streptomycin (1,000,000 units/bottle) was dissolved in 5ml of normal saline;

c. Add 1ml of penicillin and 1ml of streptomycin to 500ml of normal saline to make the final concentration 400U/ml. Mix well and store at room temperature for later use.

5.2 Primary fibroblast culture

a. Select the adult large white pigs required for the experiment (mark the sex and ear number for easy search and record), disinfect with iodine and 75% alcohol in turn, and cut off the soybean-sized tissue block band with surgical scissors (or ear size pliers) Back to the laboratory, pig ear wounds were disinfected;

b. Place the ear tissue in a petri dish on the ultra-clean bench, wash it 2-3 times with the tissue block lotion, and scrape off the hair and surface cuticle on the tissue block with a scalpel;

c. Place the tissue block in a new petri dish, wash it with cell washing solution for 3-4 times, then place it in a sterilized 25ml beaker, and cut it to ^1mm3 size with ophthalmic scissors;

d. Add a small amount of cell culture medium to the above tissue fragments, and then inoculate the tissue fragments into 3-4 125ml culture bottles with a spatula. Put the wall of the bottle with the fragments facing up, add 4ml of cell culture medium to the lower layer, and put After culturing in a constant temperature incubator for 6 hours, the cells were completely infiltrated with the culture medium, and cultured statically. Static culture for 48 hours without changing the medium is conducive to the adhesion of the tissue block;

e. It takes 10-15 days for primary fibroblasts to be cultured. Observe the state of the cells, and they can be frozen or passaged when they are full;

5.3 Cell passage

a. When the cell confluence reaches about 80%, discard the original culture medium;

b. Wash 2-3 times with cell washing solution (add 2ml to 125ml culture flask), discard the supernatant;

c. Add 0.25% Trypsin (1ml for 125ml culture flask) to make Trypsin completely infiltrate the cells;

d. Place the culture bottle in the incubator for 1-2 minutes and take it out. The wall of the bottle can be seen to be milky white. When the culture bottle is placed obliquely, the cells can be seen sliding to the bottom of the bottle, and the cells are spherical under the microscope;

e. Add cell culture medium at a ratio of 1:1-1:2 to stop digestion, use a 5ml pipette to blow the cells on the wall of the bottle, and then transfer the digestion solution to a 15ml centrifuge tube;

f. Centrifuge at 1000rpm for 5min, discard the supernatant;

g. Add 1ml of cell culture medium to suspend the cells, then inoculate the suspension into 2 corresponding culture flasks, inoculate 0.5ml of each bottle, add 4ml of cell culture medium (125ml culture flask);

h. Shake evenly in a cross shape, and culture in a constant temperature incubator at 37° C., 5% CO ₂ , and 100% humidity.

5.4 Transfer the expression vector into cells by electroporation.

a. Discard the original culture medium in the culture flask, add 2ml of PBS to wash the cells, add 1mL of 0.05% trypsin to digest the cells, and place the culture flask in a constant temperature incubator for 2 minutes;

b. Add 3mL cell culture medium to stop digestion, pipette evenly, transfer to 1.5mLEP tube, centrifuge at 1000r/min for 5min, discard supernatant to collect cells.

c. Add the mixed solution (82μl transfection solution + 18μl supplement solution) and 2μg of the linearized recombinant vector in step 1 into the EP tube, pipette evenly and transfer to the electroporation cup to ensure that the suspension covers the bottom of the cup without air bubbles, and cover it well Cover, put the electroporation cup into the electroporation instrument. Select the appropriate program and start the program for electroporation.

d. After the electroporation is completed, take out the electroporation cup, take a 60mm petri dish, and add 5ml of cell culture medium without double antibody. Transfer the suspension in the electroporation cup to a petri dish.

e. Place the incubator and keep it standing still for 24 hours, and replace the cell culture medium after 24 hours.

5.5 Seeding, culturing and screening cells

After culture, select a single colony with good cell density and good cell shape, and extract DNA for PCR identification after culture;

a. Design three pairs of primers according to the vector sequence to amplify the promoter region+sfat-1, sfat-1+2A+fad-2 and fad-2 regions respectively, and the fragment length is 500-700bp.

b. PCR program: 95°C, 5min; 95°C, 30s, depending on the annealing temperature of the primer, 30s, 72°C, 30s, 35 cycles; 72°C, 5min; 16°C, 10min.

c. Amplification result: use three pairs of primers to amplify respectively.

For the cells that have been identified as positive, the medium should be changed, and they can be subcultured to a 24-well plate or frozen after being overgrown, that is, fibroblasts carrying the sFat-1-2A-Fad-2 gene fragment without a selection marker.

Example 2

1. In vitro maturation and culture of porcine oocytes

1) The ovaries used in the experiment were all collected from slaughterhouses. After collection, the ovaries were placed in 35°C normal saline (containing streptomycin and penicillin), and transported back to the laboratory in the shortest possible time;

2) In order to better collect oocytes, prepare physiological saline (containing double antibody) in advance and preheat it, and carefully wash the collected ovaries 3-4 times to remove blood in the ovaries;

3) Collection of oocyte complexes: use a 10ml disposable syringe with a 12-gauge needle, the direction of the needle opening is downward, and the needle is inserted into the side tissue to collect the oocyte complexes and follicular fluid in follicles of 5-8mm;

4) Collect the collected follicle fluid into a 50ml centrifuge tube, discard the supernatant after resting for 15-20 minutes, wash 2-4 times with egg washing liquid at 38°C, carefully discard the supernatant after resting for 10 minutes each time;

5) Divide the washed follicle fluid into 60mm petri dishes, pick the oocytes with a suction pipette under a stereoscope, and transfer them to a 35mm petri dish filled with egg washing fluid to wash twice; During the process, pick oocytes with regular shape, uniform cytoplasm (even light transmission when observed under a dissecting microscope), and with more than 3 layers of cumulus cells tightly surrounded on the periphery (as shown in Figure 2), and proceed to the next step;

6) After the oocytes are washed twice in the egg washing solution (35mm petri dish), they are then placed in a well-balanced washing solution without hormones and washed twice;

7) Transfer to a four-well plate for culture: put 60-80 oocytes in each well, and culture them for 39-41 hours at 38.5±0.5°C, 5%±1% CO2, and saturated humidity to obtain mature oocytes ( As shown in Figure 3);

8) After that, the somatic cell nuclear transfer operation is performed.

The in vitro maturation rate and quality of porcine oocytes are closely related to the size of follicles. When the oocyte diameter is less than 3mm, it does not have the ability to mature in vitro. When the oocyte diameter is greater than 6mm, aging may occur after later culture. In the experiment, 676 ovaries with a weight of about 4g were collected, and follicles with a diameter of about 5-8mm were selected to extract oocytes, and finally 2080 oocytes with excellent quality were obtained for in vitro maturation and culture, and the mature oocytes were counted for 40±1h. The number of cells was 1601, and the oocyte maturation rate was 77%.

2. Recovery and cultivation of recipient cells:

When cells are not in use, they are usually stored in liquid nitrogen. The low temperature will not denature the protein, and the cells enter a dormant state in liquid nitrogen, with only a very low level of metabolism, and their energy can be provided by anaerobic metabolism. The freezing process is divided into liquid crystal state and glass state. The crystallization of the liquid crystal state has mechanical damage to the cells. The extremely low temperature of liquid nitrogen can ensure that the cells can quickly pass through the liquid crystal state to the glass state when freezing, thereby reducing the damage to the cells. . When the cells are thawed, the cells need to be quickly removed from the liquid nitrogen and thawed quickly to restore them to the growth state.

(1) The cell recovery experiment was carried out on the day of ovary collection;

(2) The embryo room needs to be sterilized in advance (UV irradiation);

(3) Take an appropriate amount of ultrapure water in a beaker and place it in a water bath at 38±1°C for incubation;

(4) Turn on the ultra-clean bench in the embryo room, and place the complete medium of the cells in a water bath at 37±1°C to preheat for standby;

(5) Quickly take out the cells from the liquid nitrogen and place them in a beaker and shake them constantly to ensure that the cells are heated evenly and completely dissolved. Remember not to submerge the cryopreservation tube into the water to avoid contamination and ensure that the cells can melt within 1 minute. ;

(6) To avoid contamination, after wiping the outer wall and opening of the cryopreservation tube with 75% alcohol cotton ball, transfer the cell cryopreservation tube to the ultra-clean workbench, use a pipette gun to draw the cell suspension and transfer it to a 15ml centrifuge tube, Add an appropriate amount of complete medium preheated at 37±1°C, and mix by pipetting;

(7) Place the 15ml centrifuge tube containing the cell suspension in the centrifuge, centrifuge at 1000rpm for 5min, and carefully draw the supernatant to the waste liquid tank with a pipette gun;

(8) Add 1mL of complete medium to resuspend the cells, gently blow and mix, put into a 24-well cell culture dish, 500μl per well (if the cell concentration is high, dilute several wells), and mark the cell name on the culture dish , time and other information, gently shake the culture dish in a criss-cross direction (to ensure uniform distribution of cells), transfer to a 37±1°C, 5%±1% _CO2 cell culture incubator for culture;

(9) The resuscitated cells need to change the medium after 24±1h. In the ultra-clean bench, gently tilt the culture dish, use a pipette gun to absorb the medium and transfer it to the waste liquid tank, and then wash it with DPBS 1-2 times. Then slowly add 500 μl complete medium;

(10) When waiting for the cells to grow for about 16 hours, observe the cell growth under a microscope, and digest the cells when the cell density is about 78%-82%;

(11) Aspirate the medium in the ultra-clean bench, rinse 2-3 times with preheated DPBS, add trypsin with a concentration of 0.025% for digestion, and observe under the microscope that most of the cell edges are upturned into balls, and the trypsin Aspirate the enzyme, add 100-200ul of complete culture medium to stop digestion, add 1ml of H199 operating solution to blow up the cells on the wall, transfer to a 15ml centrifuge tube, observe under the microscope whether the cells on the wall are completely blown off, after repeated blowing and blowing, add more H199 operating solution to 6ml, 1000rpm, centrifuge for 5min, discard the supernatant (pour out as much as possible), resuspend the cells with a 100ul gun after the culture fluid retained on the tube wall flows down, transfer to a 1.5ml EP tube, and transfer the cells into the tube with a 10μl gun. Transfer the nuclear operation and drop it for use.

3. Enucleation and nuclear transfer of mature oocytes

(1) Select mature oocytes; use a 4×10 times stereomicroscope to observe, and select oocytes that are in good growth condition and discharge the first polar body for subsequent use;

(2) Mature oocytes need to be treated in advance: add the hyaluronidase solution into the mature oocytes, and repeatedly blow and beat, the purpose is to remove the cumulus cells;

(3) Install the holding needle on the left, and install the coring needle on the right; before installing the needle, the coring needle must be exhausted, and the paraffin oil should be filled when it is less than 2/3 of the length of the operating arm, and the needle bevel is opposite to the operator. ;Adjust the holding needle and injection needle to make them level;

(4) Make a 100mm culture dish cover, use operating solution H199 (with CB (5 μg/ml)) for the enucleation plate to make long droplets, and then cover with paraffin oil; make three long droplets for the nuclear transfer plate, and the middle one For operation (with CB), somatic cells are placed on the left and right sides (without CB);

(5) Adjust the field of view: immerse the operating needle in the operating solution, observe from low magnification to high magnification, and finally operate under a 40× microscope;

(6) Enucleation operation: under a 40× microscope, adjust the height of the holding needle so that it is slightly higher than the oocyte. After absorbing an oocyte, adjust the direction of the oocyte with the injection needle so that the holding needle is at At nine o’clock, the first polar body is at three o’clock, adjust the height of the enucleating needle so that it is at the same focal length, enter the needle from the three o’clock direction, remove the cytoplasm at about 1/4, and exit the needle When the first polar body is brought out;

(7) Nucleus transfer: The processed oocytes are transferred to the operation drop bar, and the injection needle is placed in the cell operation drop. The injection needle has been inhaled to select a medium size, strong refraction, round shape, dense cytoplasm, and good vitality. recipient cells (about 100-200).

Hold the fixed needle under all the oocytes, give the force to suck the oocytes tightly, after sucking the oocytes, adjust the focal length so that the oocytes and the injection needle are on the same plane, and give the injection needle a little bit outward to receive The force of somatic cells, when the recipient cells move to the injection needle, quickly penetrate the perivitelline space of the oocyte, and quickly remove the injection needle after the recipient cells enter the oocyte;

After the nucleus transfer is completed, use the injection needle to move the reconstituted oocyte downward, and the upper oocyte will be automatically sucked tightly by the holding needle, and the nucleus transfer operation will be repeated until all are completed. The operation process is shown in Figure 4.

(8) Remove the completed reconstituted egg cells and wait for fusion in the H199 operating solution. If not used temporarily, wash several times in the H199 operating solution, then wash three times in the embryo culture solution and transfer to the embryo transfer nucleus The culture medium is used in culture drops for later use.

In the process of enucleation and nuclear transfer, the selected nuclear transfer cells should be as uniform in size as possible so that they can be evenly arranged in the injection needle in order to achieve a better transplantation effect; Do not inhale the liquid into the operating arm. If it is inhaled, it should be cleaned with absolute ethanol in time, and it can be used after it is completely volatilized.

4. Parthenogenetic activation and fusion of reconstituted oocytes

(1) The activation tank is wiped and disinfected with alcohol before use, and it is ready for use after being exposed to ultraviolet light;

(2) The activation solution and operating solution should be preheated in a water bath at 37±1°C.

(3) Prepare three PZM-3 culture plates, and equilibrate for about 4 hours at 38.5±0.5°C and 5%±1% _CO2 for later use;

(4) Connect the fusion tank and the electrodes, make sure the circuit is unobstructed, and adjust the parameters of the fusion instrument: 2.0kv/cm, 50us;

(5) Clean the fusion tank, and repeatedly blow and blow near the electrode with the tip of the pipette to remove air bubbles;

(6) Take about 50 reconstituted oocytes each time and put them into the H199 operating solution and wash them 1-2 times;

(7) Put it in the activation solution to balance for 1-3 minutes, and take it out quickly when the oocyte sinks after putting it in;

(8) Put the activation solution in the tank, and after preheating twice with air shock, put the oocytes into the activation tank;

(9) Adjust the position of the oocyte with the egg-pushing needle, so that the contact surface between the donor cell and the oocyte is perpendicular to the direction of the current, and shock once;

(10) Remove after electric shock, place in H199, wash three times, transfer to embryo culture solution and rinse four times, put in 38±0.5°C, 5%±1% _CO2 incubator for cultivation, and calculate the fusion rate;

The total number of enucleated oocytes was 751, the total number of donor cells transferred was 600, the total number of fusion was 560, and the fusion rate was 93.3%.

In the experiment, the morphology of cell division 24h, cell division 48h, and blastocyst 6d were respectively recorded, as shown in Figure 5-7. According to the test statistics, the 48h cleavage rate was 53.7%, and the 7d blastocyst rate was 29.1%.

Example 3

pig embryo transfer

(1) Surgical instruments need to be sterilized in advance: Mitsubishi suture needles, circular suture needles, scalpel handles, blades, hemostatic forceps, needle holders, etc. need to be sterilized at 160°C and stored aseptically for later use; sutures and gauze need 110kPa , sterilized at 120°C for 30 minutes, and also aseptically dried and stored for later use;

(2) Select healthy Landrace sows with normal estrus cycle for 2-3 consecutive times as transplant recipients, and control the estrus time within 48 hours;

(3) Surgical method is used for embryo transfer. After the recipient sow is anesthetized, one side of the uterus is pulled out, and a blunt needle is used to pierce the uterine horn at a distance of about 7 cm from the tip of the uterine horn to perform embryo transfer, and then follow the routine Surgical treatment of the wound and careful care to avoid wound infection. At this time, the sow needs to be raised separately; the transgenic pig will be obtained after calving.

Example 4

Identification of transgenic pigs-DNA level

1. Extraction of positive plasmids

(1) Take out the seed-preserving bacteria solution from the -80°C refrigerator, transfer 20 μl of the bacteria solution to a 50ml centrifuge tube with a pipette gun, then add 15ml of LB medium and 15μl of ampicillin, cover and seal it, and place it on a constant temperature shaker , keep 200rpm, incubate at 37°C for 8 hours, observe the turbidity of the bacterial solution;

(2) After 8 hours, take out the centrifuge tube, and stop the culture when the bacterial solution becomes turbid, then extract the plasmid according to the instructions of PlasmidMiniKitⅠ (OMEGA) for extracting the plasmid; then use 0.8% agarose gel to identify the extraction result, as shown in Figure 8 .

2. Extraction of DNA

Take a mung bean-sized ear tissue sample, put it into a 1.5ml centrifuge tube, and cut it into pieces. Use the Biotek Tissue Genomic DNA Extraction Kit to extract the whole genome DNA from the ear tissue of 5 pigs. The DNA extraction results are shown in Figure 9.

3.PCR amplification primers

The primers were synthesized by Yingwei Jieji Company. The upstream and downstream primer sequences of sFat1 are shown in SEQIDNo.4 and 5 (the target fragment is 610bp); the upstream and downstream primer sequences of the 2A sequence region are shown in SEQIDNo.6 and 7 (the target fragment is 511bp) ; Fad2 upstream and downstream primer sequences are shown in SEQ ID No.8 and 9 (the target fragment is 591bp).

PCR system and conditions

PCR 50 μl system: 2×TaqMasterMix 25 μl; 2 μl each of upstream and downstream primers (10 μM), template less than 1 μg, filled with double distilled water;

PCR reaction conditions: pre-denaturation at 95°C for 5min; denaturation at 95°C for 30s, annealing for 30s, extension at 72°C for 45s, a total of 34 cycles; extension at 72°C for 5min. Among them, the annealing temperature of sFat-1 was 57.5°C; the annealing temperature of 2A was 59°C; the annealing temperature of Fad-2 was 57°C.

The PCR amplification product is detected by 1.5% agarose gel electrophoresis, and a 100bp marker is selected. During electrophoresis, the voltage is kept at 100-120V for about 25 minutes, and the target gene amplification results can be recorded in the gel imager. , as shown in Figure 10.

The positive plasmid and the PCR amplification products of the three pairs of primers of the sample DNA were all sent to Zhongmei Taihe Company for sequencing, and the sequencing results were compared with the carrier sequence, and the sequence was correct.

4. Identification of RNA levels

4.1 Extraction of RNA

The Biotech kit was used to extract total RNA from tissue samples of piglet heart, liver, spleen, lung, kidney, stomach, intestine, back muscle, and leg muscle. The agarose gel electrophoresis of the extracted RNA is shown in Figure 12. It can be seen from Figure 11 that the extracted RNA is intact.

4.2 Synthetic cDNA and expression analysis of foreign genes

All cDNA synthesis was carried out in accordance with the operating instructions of the Thermo reverse transcription kit. The total amount of RNA was 500ng. Reagents such as oligodT, Buffer, DNTPs, DTT, and reverse transcriptase were all added in accordance with the recommended amounts in the instructions. The PCR program was set at 65°C. 5min; 42°C, 1h; 70°C, 10min; During the operation, note that the RNA should be stored on ice at low temperature to prevent degradation. The EP tubes used in the reverse transcription process are RNase-free centrifuge tubes, and the It is carried out in the ultra-clean bench, and the EP tube needs to be carried out on a low-temperature metal bath.

4.3 QPCR primer design

The cDNA sequence of the exogenous gene was used as a template, and the primer5 software was used to design primers. At the same time, the internal reference gene GAPDH primer was designed and synthesized by Yingwei Jieji. The upstream and downstream primer sequences of sFat1 are shown in SEQIDNo.10 and 11 (the target fragment is 216bp); Fad2 The sequences of the upstream and downstream primers are shown in SEQIDNo.12 and 13 (the target fragment is 179bp); the sequences of the GAPDH upstream and downstream primers are shown in SEQIDNo.14 and 15 (the target fragment is 233bp).

4.4 QPCR system and conditions

PCR 20μl system: 2×SYBRSelectMasterMix 10μl; upstream and downstream primers (10μM) each 0.8μl, template cDNA (dilute the original solution uniformly 50 times) 2μl, double distilled water to make up.

GraphPad Prism6 statistical analysis software was used for analysis, and Adobe Photoshop CS6 drawing software was used for processing. The results are shown in Figure 12. Statistical analysis results showed that the expression of exogenous gene could be detected in different tissues such as heart, liver, spleen, lung, kidney, stomach, intestine, back muscle and leg muscle in transgenic piglets, but the expression level was low. The expression of both genes was not detected in piglets in the control group.

While particular embodiments of the invention have been illustrated and described, it should be appreciated that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims (10)

1. a method for preparing a multigene compound character genetically engineered pig without selection marker, is characterized in that, comprises the following steps: Using fibroblasts carrying sFat-1-2A-Fad-2 gene fragments without selectable markers as donors and enucleated mature porcine oocytes as recipients, the somatic cell nuclear transfer method was used to obtain cells containing non-selectable markers Reconstructed embryos of polygenic compound trait genes; After the reconstituted embryo is cultured in vitro, it is transplanted into a surrogate pig, and after the pig is delivered, a genetically engineered pig with multiple genes and complex traits without selection markers can be obtained. 2. The preparation method according to claim 1, characterized in that the oocytes are extracted from follicles with a diameter of 5-8mm, and the oocytes are matured in vitro to obtain mature porcine oocytes. 3. preparation method according to claim 2, is characterized in that, described oocyte carries out in vitro maturation culture and obtains mature porcine oocyte specifically: The oocytes were cultured at 38.5±0.5° C., 5%±1% CO ₂ , and saturated humidity for 39-41 hours. 4. preparation method according to claim 1 is characterized in that, the oocyte that the mature porcine oocyte of described enucleation adopts is the oocyte that grows well and discharges the first polar body; And the The above-mentioned oocytes are blown repeatedly with a hyaluronidase solution before enucleation to remove cumulus cells. 5. The preparation method according to claim 1, characterized in that the recovery time of the frozen donor cells is carried out on the day of ovary collection; When resuscitating cryopreserved donor cells, quickly remove the cells from liquid nitrogen and place them in a water bath at 38±1°C with constant shaking, and the cells will melt within 1 min; Transfer the thawed cells to a centrifuge tube and add 37±1°C preheated complete medium, pipette and mix well, then centrifuge, the obtained cells are resuspended, inoculated into 37±1°C preheated complete medium for culture, culture After 24±1 hours, wash 1-2 times with DPBS, and then change the medium once; After culturing until the cell density reaches 78%-82%, the cells are digested, and then the cell nucleus transfer operation can be performed. 6. The preparation method according to claim 5, characterized in that, the method of somatic cell nuclear transfer is specifically: Mature oocytes are enucleated to remove the first polar body, and then transferred to the operating drop bar; Select recipient cells with medium size, strong refraction, round shape, dense cytoplasm, and good vitality, inhale into the injection needle, and place the injection needle in the cell operation drop; Hold the fixed needle and place it under the enucleated oocyte, and give the force to suck the cell tightly. After the cell is sucked tightly, adjust the focal length so that the enucleated oocyte and the injection needle are on the same plane, and slightly push the injection needle outward The force of the recipient cells, when the recipient cells move to the injection needle, quickly penetrate the perivitelline space of the oocyte, and quickly remove the injection needle after the recipient cells enter the oocyte to obtain the reconstructed oocyte; The reconstituted egg cells are activated and fused, and cultured to obtain reconstituted embryos. 7. The preparation method according to claim 6, characterized in that, the activation of the reconstituted oocytes specifically comprises: putting the reconstituted oocytes into the H199 operating solution, washing them for 1-2 times, and putting them into the activation solution to equilibrate for 1 -3min, when the reconstituted egg cells sink and take them out quickly; Put the activation solution in the fusion tank, and after warming up twice with air shock, put the reconstituted egg cells into the activation tank; The H199 operating fluid and the activation fluid are preheated before use. 8. The preparation method according to claim 7, wherein the fusion is performed by electric shock; The electric shock is performed by a fusion instrument, and the parameters of the fusion instrument are: 2.0kv/cm, 50us. 9. The preparation method according to claim 8, wherein the fusion specifically comprises: adjusting the direction of the reconstructed oocyte so that the contact surface between the donor cell and the oocyte is perpendicular to the direction of the current, and shocking once; Remove after electric shock, place in H199, wash three times, move to embryo culture solution and rinse 3-4 times, put in 38±0.5°C, 5%±1% CO ₂ incubator for cultivation. 10. The preparation method according to any one of claims 1-9, wherein the surrogate pig is: a healthy Landrace sow with 2-3 consecutive estrus cycles, and the estrus time is controlled within 48 hours Inside.