Research Of CRISPR/Cas9 Knockout MSTN Genein Ovis Aries Fibroblasts

Objective: Myostatin, or growth and differentiation factor 8(GDF8), a member of the transforming growth factor β(TGF-β) family, and it was proved to be negative regulatory factor to the growth of skeletal muscle. Loss of the function of this gene is associated with the phenotype described as “double muscling”, an extreme form of muscle development characterized by a large increase in muscle mass. Two kinds of latest design artificial nuclease can be used for various complex genome editing CRISPR/Cas9 and TALEN are based on the use of engineered nucleases composed of sequence-specific DNA-binding domains fused to a nonspecific DNA cleavage module. The experimental design CRISPR/Cas9 and TALEN respectively in Ovis aries MSTN gene exon 1 and exon 3. To transfect CRISPR/Cas9 and TALEN plasmids into Ovis aries fibroblasts, in order to achieve the Ovis aries MSTN gene mutation, lay the foundation for CRISPR/Cas9 and TALEN plasmids on MSTN gene carrying out feasibility studies.Methods:1. “Golden Gate” cloning method to achieve building CRISPR/Cas9 and TALEN plasminds. Transformation and extraction of plasmid, agarose electrophoresis detecting whether successful build of plasminds. 2. Using the L- solution formulated in our lab, p MAX plasmid expressing Green Fluorescent Protein(GFP) was electroporated into sheep fibroblasts by Lonza 4D- Nucleofector. Transfection efficiency was based on the measurement results by flow cytometry and fluorescence microscopy, different electrotransfection programs, number of cells, plasmids concentration, number of electric shock and temperture after transfection, an ideal transfection efficiency could be obtained. 3. Identification of activity of CRISPR/Cas9 and TALEN plasmids: The ovine MSTN gene first exon with CRISPR/Cas9 and third exons with TALEN were electroporated into Ovis aries fibroblasts. Surveyor nuclease, T7E1 and HRM three methodscan be used to determine the purpose of DNA mutations. 4. Screening of monoclonal mutations in the cell: Different individual cell are obtained by the cultivation of a single cell. Use of HRM and PAGE methods to screening monoclonal cells, get MSTN gene mutation cell line, obtain monoclonal mutation cells.Results: The experimental 1 results show that: Successfully Construction of CRISPR/Cas9 and TALEN plasmids. Agarose electrophoresis detecting results show that Cas99999 bp, sg RNA 4930 bp, TALENL/R 8322 bp. The experimental 2 results: The results showed that cells were pelleted and resuspended in 100μl L-solution at a concentration of 2×106 cells and electroporated using the nucleofection program CZ-167, 4μg plasmid DNA, at 37℃ for ten minutes after transfection, an ideal transfection efficiency could be obtained. The experimental 3 results: Three methods of detection of artificial nuclease biological activity, Surveyor nuclease, T7E1 and HRM detecting the purpose of DNA mutations.Results is sheep MNST gene exon 1 CRISPR/Cas9 and exon 3 TALEN goal targets are mutated. The experimental 4 results: The test using HRM and PAGE detection mutation, mutated cells directly sequenced, by comparing the sequencing results we obtained the mutation monoclonal sheep fibroblasts.Conclusion: 1. Successful build of plasminds CRISPR/Cas9 and TALEN plasmids respectively in Ovis aries MSTN gene exon 1 and exon 3. 2. Electric transfection CRISPR/Cas9 and TALEN plasmids into Ovis aries fibroblasts, through Surveyor nuclease, T7E1 and HRM three methods can be used to determine CRISPR/Cas9 and TALEN plasmids have biological activities and cutting goal targets. 3. Different individual cell are obtained by the cultivation of a single cell. Use the HRM, PAGE and direct sequencing three methods to screening monoclonal sheep fibroblasts.