Editting Rabbit Fibroblast Myostatin Gene In Cas9 Technology, Target Selection And Identification

The myostatin gene also called Growth differentiation factor 8, belongs to the TGF-βsuperfamily as a negative regulator of skeletal muscle growth. Mutations occurred in the conservative regions of myostatin gene may affect even cause the loss of its function of muscle inhibiting, thereby causing celluar hyperplasia and hypertrophy of the skeletal muscle, may also affecting growth triats and performance triats in a range of mammalian spieces.Clustered regularly interspaced short palindromic repeat sequences( CRISPRs) system can cut foreign sequences, which is a RNA-directed system modificated gene technology.Our experiment based on CRISPR/Cas9 technology, Taking rabbit fibroblasts myostatin gene as target to edite, We selected a target in mature peptide of rabbit myostatin exon. converted bacterial Cas9 cDNA into rabbit Cas9 cDNA by optimizing mammalian codons,connected it to pUC57 plasmid, and then transfected Cas9 and gRNA with different ratios of moles, the 7 gradient respectively is 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, extracted genomic DNA from fibroblasts, then PCR, hybridization, T7E1 digestion, At the end of these treatments, we measured the targeting efficient. Then we screen monoclonal cells in the 2:1 experimental group, selected 15 monoclonal cells for further incubated. After that, we extracted the genome, PCR and connected PCR products to T-vector, sequencing, and results showed that it have gene mutation and deletion, and the alignment amino acid also have mutations and shifts. Identify the expression situation of Myostatin gene by Western blot, two in five base mutant cells was knock-out from the Myostatin cell strains.Using CRISPR/Cas9 technology, we successfully edited rabbit fibroblasts myostatin gene, and screen knock out myostatin gene fibroblast strains, laid the foundation of follow-up living body experiments.