Method for knocking out bovine myostatin gene by zinc finger nuclease
A myostatin and zinc finger nuclease technology, applied in the field of genetic engineering, can solve the problems of inability to obtain positive single cell clones, high risk, and long cycle, so as to simplify the biosafety evaluation process, save the drug screening process, and avoid Anti-drug effects
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Embodiment 1Z
[0031] Screening and knockout efficiency of embodiment 1 ZFN expression vector
[0032] 1. ZFN expression vector construction
[0033] MSTN (NC 007300.4) gene sequence information was obtained from the NCBI website, ZFNs design was completed by Sigma Company, and the designed ZFNs sites were located on exons 1 and 3. MSTN-ZFNs-Set 1 acts on the first exon, and MSTN-ZFNs-Set 2 and MSTN-ZFNs-Set 3 act on the third exon. The DNA sequences they act on are:
[0034] ZFNs-Set1: GTCATTACCATGCCCACGG AGTGTG AGTAGTCCTGCTGGT ;
[0035] ZFNs-Set 2: CTCATCAAACCCATGA AAGACGG TACAAGGTATACTGG ;
[0036] ZFNs-Set 3: TTCCCAGAAC CAGGA GAAGATGGACTGGTA
[0037] The underlined parts are zinc finger protein binding sequences, and the middle part is the FokI endonuclease cleavage site. The corresponding three ZFNs expression vectors are: MSTN-ZFN-Set1-pZFN1 / pZFN2, MSTN-ZFN-Set2-pZFN1 / pZFN2 and MSTN-ZFN-Set3-pZFN1 / pZFN2.
[0038] ZFN expression vector construction process: Taking MST...
Embodiment 2
[0045] Example 2 Obtaining of Single Cell Clones and Identification of Gene Knockout Clones
[0046] 1. Obtaining of single cell clones
[0047] Using the AMAXA electroporation instrument to electrotransfer bovine fibroblasts, using optimized electrotransfer parameters T-016, the gene transfection efficiency can reach more than 90%. The transfected genetic material is mRNA, and the half-life in the cell is about 8 hours. There will be no random insertion into the cell genome when the DNA is transfected, which has a good guarantee for the genetic stability of the animal. At the same time, there will be no random integration of resistance genes, which meets the requirements of biological safety.
[0048] Specific operation method: Recover and purify the in vitro transcribed mRNA with a kit from Applied Biosystems, elute and dissolve with DEPC water, and make the final concentration about 500 ng / μl. The mRNA corresponding to a pair of ZFNs is 2 μg each, the total mRNA amount is...
Embodiment 3
[0052] Example 3 Preparation of embryos of gene knockout single-cell clones and cloned cattle
[0053] 1. Preparation of knockout cattle
[0054] The specific process includes:
[0055] (1) Holstein cow fetal fibroblast culture
[0056] Fetal ear tissues of 40-day-old Holstein cows were taken, and the bovine fetal fibroblast cell line was established through primary culture, subculture, freezing and other in vitro culture operations.
[0057] (2) Gene knockout single cell clone
[0058] The acquisition of gene knockout single cell clones is the same as in Example 2.
[0059] (3) Transgenic cloned embryo preparation and embryo transfer
[0060]Collect the ovaries of adult cattle from the slaughterhouse, take follicles with a diameter of 2-8 mm, and recover the cumulus-oocyte-complex with uniform shape and compact structure, and divide the cumulus-oocyte-complex into 50-60 pieces Put each hole into a four-well plate containing maturation solution (M199+10% fetal bovine seru...
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