转基因小鼠 z-bo 2020-10-14 374

　　(1) The selection of donor mice, excessive ovulation and reproduction should be based on the purpose of constructing transgenic animals to determine the type and pedigree of donor mice. To create an animal model, it usually has a clear genetic background, a high degree of homogeneity (that is, the degree of genetic identity of each body in the lineage) and homogeneity (the homogeneity of each body at all gene loci) . Strains with more scientific characteristics and ovulation number), and choose high-quality embryos. For example, C57BL, Paramouth, etc. Generally, 5-6 weeks old, 18-22 g, healthy, sexually mature SPF female mice are used as donors. Determine the number of donors according to the experimental scale.

　　After the donor, the lighting of the animal room was adjusted from 6 am to 6 pm during the day and from 6 pm to 6 pm at night. PMSG5-10IU is injected through the tail vein or intraperitoneal (usually 13:00-14:00), and HCGj-10IU is injected 46-48 hours later. After HCG injection, normal male mice (1:1) were placed in the cage, and the sitting posture was confirmed in the early morning of the next day. One with a vaginal sitting position is a fertilized egg donor mouse.

　　(2) Select recipient mice and prepare false pregnant female mice. Recipient mice usually choose the same strain as the donor. However, like surrogate female mice, other strains with strong fertility and good maternity can also be used. Generally, healthy female mice weighing more than 25 g and aged 7-8 weeks are used as recipients. To be prepared

　　fake pregnant female mice, please obtain ligated male mice first. Male rats aged 6-8 weeks are anesthetized and undergo abdominal surgery. Ligate, suture and rest the incisions on both sides for at least two weeks. On the same day that the donor mice were housed, the recipient female mice were housed together with the ligated male mice (1:1). On the second day, the vaginal seat was examined and it was a pseudo-pregnant female mouse with a vaginal seat.

　　(3) Obtain fertilized eggs. Collect pronuclear embryos in the afternoon when the donor mice are tested with vaginal sac. The donor mouse was decapitated, the Faropius tube was cut in a sterile environment, and placed in a 200μl M2 drop, pierced with an ampoule under a solid microscope, and gently squeezed the coated o-stone cells, 300μg/ml hyaluronic acid Enzymatic fertilized eggs are digested with M2 solution. After the granuloma cells fall off, the fertilized egg is washed 3 times with M2 droplets. During this process, the quality of the fertilized eggs was checked, and unfertilized eggs and other abnormal eggs were eliminated. The normal eggs were transferred to M16 solution and placed in an incubator at 37°C and 5% CO2.

　　(4) Gene injection into a miniature manipulator. The inverted microscope must have a phase difference system or a differential interference contrast system. The egg fixing needle is installed on the left and the injection needle is installed on the right to adjust the length of the lens and focus. Place a drop of ophthalmic culture medium on the center of the concave glass slide, transfer 10 to 20 fertilized eggs, and cover with mineral oil. Under the micromanipulator, use a fixed needle to suck out the fertilized egg, insert the injection needle containing DNA into the male pronucleus of the fertilized egg, and inject the DNA solution (about 2 pL, containing about 500-600 gene copies). .. After seeing a slight expansion of the male anterior nucleus, he immediately pulled it out of the needle. Some data suggest that the injection of two pronuclei can increase the integration rate, so the female pronucleus can be injected in the same way. After all the fertilized eggs were injected, they were placed in another droplet containing 80 μl of M16 medium and placed in a 5% CO2 incubator at 17°C.

　　(5) Transplanted pseudo-pregnant mice were anesthetized with sodium pentobarbital at a dose of 100 ms/kg body weight by tail vein injection or Smianxin II (10 mice each diluted, 0.2 ml each). )) Hair loss by intraperitoneal injection, a small opening of 0.5 cm is cut in the new part, avoiding the blood vessels, and leading out the Faropius tube and ovaries. Under a solid-state microscope, the transfer needle sucks the fertilized egg injected with 20-30 DNA in the M2 solution, and then inserts the fibril of the fallopian tube to transfer the fertilized egg to the ampla of the fallopian tube. Then the Faropius tubes and ovaries were put back into the pseudo-pregnant mice, sutured and disinfected. The entire process should be completed as soon as possible to reduce the negative effects of exposure to eggs. After completion, the recipient mice were sent to the risk cage to strengthen management and observation.

　　Bubble 3 is to prevent the eggs from falling when the egg transplant needle is not inserted into the transplant site, while Bubble 2 and Bubble 1 are to ensure that all eggs are moved to the ampla. it is.

　　(6) Integration and expression detection

　　After the recipient mouse is born, the tail of the offspring is cut off and DNA is extracted. First perform PCR detection, and then perform Southern hybridization with PCR-positive samples to confirm transgenic mice. According to the type of protein expressed, radioimmunoassay, fluorescence immunoassay, ELASA, Weathern blot and other techniques are used to detect the expression of transgenic mice. Some people need to test the corresponding function.