A kind of expression vector and application of porcine muscle specific expression follistatin-317
An expression vector and specific technology, applied in the field of genetic engineering, can solve problems such as reproductive obstacles and hidden safety hazards of transgenic pigs, and achieve the effect of increasing safety.
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Embodiment 1
[0035] Embodiment 1 vector construction
[0036] see figure 1 , using Red / ET-mediated homologous recombination, the first step is to capture the 21kb locus containing porcine skeletal muscle actin onto the capture vector; the second step is to knock in the FST317 gene plus the resistance gene into The coding region of skeletal muscle actin; the third step is to delete the resistance gene through Flpase-mediated site-specific recombination, and the vector construction is completed.
[0037] 1.1. Capture carrier construction
[0038] The left and right homology arms were amplified by PCR and inserted into the pBR322-loxp-neo-loxp backbone vector to construct a capture vector.
[0039] Using BAC containing porcine skeletal muscle actin as a template, respectively:
[0040] TYB1F: 5′-CGTaagcttAAATCACTGGCTGTGTGCTG-3;’
[0041] TYB1R: 5'-CTGCAGGCTGAATTCTTTCC-3';
[0042] TYB2F: 5'-TCGgaattcCTGAAA-ATTCCCCAAGACGA-3';
[0043] TYB2R: 5'-CAGtctagaGAATGGTGGAGGCGAATAGA-3';
[0044]...
Embodiment 2
[0074] Embodiment 2 transgenic cell obtains
[0075] 2.1 Establishment of Duroc pig fetal fibroblasts
[0076] 2.1.1. Take a Landrace pig that has been pregnant for 30 days, take the oviduct and uterus, bandage the exit, and transport it back to the laboratory within 2 hours. Take out the fetus from the uterus, wash the fetus with DPBS containing antibiotics, transfer it to the ultra-clean workbench, remove the head, limbs and internal organs of the fetus with ophthalmic scissors, and rinse with DPBS;
[0077] 2.1.2. Use ophthalmic scissors to cut the remaining part into pieces as much as possible in a cell culture dish with a diameter of 100mm;
[0078] 2.1.3. Add a little serum, cut off the tip of the 1ml gun tip with scissors, leaving a part with a diameter of about 40mm or more, connect the 1ml gun, transfer the tissue pieces to the bottom wall of three T25 cell culture flasks, and use an elbow pipette Spread the tissue block evenly;
[0079] 2.1.4. Turn the side covere...
Embodiment 3
[0086] The preparation of embodiment 3 transgenic pigs
[0087] 3.1 In vitro maturation of porcine oocytes
[0088] Take the ovary from the slaughterhouse, put it in 28°C-35°C normal saline containing penicillin and streptomycin sulfate, and transport it back to the laboratory within 2 hours. Use a 20mL syringe equipped with a 18-gauge needle to aspirate the 3-6mm follicles on the ovary. . Put the extracted solution in a 50mL centrifuge tube, let it stand in a water bath at 7°C for 15 minutes to remove the supernatant, add PVA-TL-HEPES to resuspend the precipitate, and let it stand for 15 minutes, repeat once, and put the resuspension into a plastic petri dish with a diameter of 60mm , under a stereomicroscope, use a mouth pipette to select cumulus-oocyte-complexes (Cumulus-Oocyte-complexes, COCs) with more than two layers of cumulus, which are dense and uniform in cytoplasm, and washed with mature culture medium for 3 Transfer to culture droplets that have been equilibrated...
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