CRISPR/Cas9 System-mediated CSN2 Gene Knock-out For Preparation Of Goat Fetal Fibroblasts Mutation Cells

Animal mammary gland bioreactor,as a technology for producing recombinant protein,has important application value in mass production of recombinant protein.However,Integrate exogenous gene into Animal genome by gene editing technology,the expression of recombinant protein in milk of transgenic animals is always at a low level.CRISPR/Cas9 has been applied to many areas of research,for its simple design and efficient editing.The study using CRISPR/Cas9 system to construct knockout vector of goat fetal fibroblast CSN2 gene,Obtain high efficiency sg RNA and knockout mutant cell lines for the material CSN2 gene knockout goats,Moreover,Investigate the efficacy of CRISPR/Cas9 system in knockout of CSN2 gene in fetal fibroblasts of goat by using double carrier transfection,In addition,the proportion of the highest positive cells was selected to obtain the program,According to the combination of different label carriers,the results are as follows:1.five sg RNA were designed and synthesized in CSN2 gene second and eighth exons of goat fetal fibroblasts,which were successfully constructed(sg RNA T1?sg RNA T2?sg RNA T3 ? sg RNA E1?sg RNA E2);the result show that sg RNA T1 and sg RNA E2 knockdown efficiency up to 34.9% and 25.9% by T7 EI enzyme digestion;selected monoclonal green fluorescent cell to culture,the result of sequencing and Western blot show that successfully obtained two strains of CSN2 gene mutant cell lines.2.Optimization of dairy goat fetal fibroblast F4 cells transfection conditions,Using 5 ? g PX330-e GFP and 10 ? l Lipofectamine 2000 plasmid transfected cells,which ranged from 81 ~ 90% for 6h,cell transfection efficiency reached a maximum of 24.3%?3.The recombinant plasmid of e GFP-sg RNA T1 and Neo-sg RNA E2,e GFP-sg RNA T1 and e GFP-sg RNA E2 were cotransfected into goat fetal fibroblast cells,monoclonal sequencing results showed that knockdown of length up to 7760 bp,the knockdown efficiency up to 16.2% by T7 EI enzyme digestion,the results show that the e GFP+Neo group get a high proportion of long fragment mutant cells(P<0.05)by PCR and immunofluorescence quantitative fluorescence detection.