Method for knocking out beta 6 subunit genes of bovine integrins by utilizing zinc finger nucleases (ZFNs)
A zinc finger nuclease and integrin technology, applied in the field of genetic engineering, can solve the problems of long cycle, high cost, low targeting efficiency, etc., and achieve the effects of promoting the formation, improving the development rate and improving the preparation efficiency.
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Embodiment 1
[0036] Example 1: Construction of ZFN expression vector and test of its gene knockout function
[0037] 1 Construction of ZFN expression vector
[0038] According to the sequence information of integrin β6 subunit gene (NM174698), the design of ZFNs was completed by Sigma Company. The sequence of the β6 subunit gene recognized by the designed pair of ZFNs (uppercase letters) and spliced (lowercase letters) is as follows: TGTTCTTTCTATGTCtaggaaGGAATGATCACGTACAAG, as shown in sequence 3 in the sequence listing.
[0039] The structures of the ZFN recombinant expression vectors pBeta6-ko-ZFNP1 and pBeta6-ko-ZFNP2 that can recognize and cut the integrin β6 subunit gene are as follows: figure 1 A. figure 1 As shown in B, the backbone of the vector is pAVX, which contains pUCori, CMV promoter, BGHpA and kanR genes. According to the presumed coding sequence of zinc finger protein that can specifically bind to integrin, EcoRI-Flag-NLS-ZFNP1-BamHI and EcoRI-Flag-NLS-ZFNP2-BamHI gene...
Embodiment 2
[0049] Example 2: Obtaining monoclonal cells knocked out of the integrin β6 subunit gene
[0050] 1 Monoclonal culture of transfected pBeta6-ko-ZFNP1 / pBeta6-ko-ZFNP2 cells
[0051] The linearized pBeta6-ko-ZFNP1 and pBeta6-ko-ZFNP2 recombinant plasmids were transfected into bovine fetal fibroblasts, the cells were counted after 24 hours, and the cells were seeded in a 10cm culture dish according to the amount of 500 cells per dish, using 15% DMEM medium with FBS at 37°C, 5% CO 2 After 1 week of culture under certain conditions, observe under a microscope and mark the monoclonal cells in a good growth state with a marker pen. Using the method of cloning rings and trypsin digestion, the monoclonal cells are expanded to 48-well plates for culture. After the cells covered the monolayer, the cells were digested with 0.25% trypsin, and 10% of the cells were taken out for PCR amplification with primers P1 / P2 to identify whether gene knockout occurred. In addition, 90% of the cells ...
Embodiment 3
[0054] Example 3: Preparation of Integrin β6 Subunit Gene Knockout Transgenic Embryos and Cattle
[0055] 1 Obtaining of gene knockout monoclonal cells
[0056] Using method 2 of Example 2, cell clones with double knockout of the integrin β6 subunit gene with frameshift mutation were obtained, and the analysis results of gene deletion and insertion were as follows Figure 5, as nuclear donor cells for the production of transgenic embryos and cattle.
[0057] 2 Mature culture of oocytes
[0058] The ovaries of adult dairy cows and cattle from the slaughterhouses in the surrounding area were washed with PBS three times, and the follicles were extracted with a needle with a diameter of 0.7mm, and the cumulus-oocyte-complex with uniform shape and compact structure was recovered. It was washed twice with maturation solution (10% FBS, 0.01U / mLbFSH, 0.01U / mLbLH and 1 μg / mL estradiol added to M199 culture solution), and then the cumulus-oocyte-complex was washed at 50- Put 60 piece...
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