Study On CRISPR/CAS9 Mediated Knock-In Of Human INS Gene In Bovine Fetal Fibroblast

Mammary gland bioreactor is a transgenic technology which is used to drive exogenous gene expression by specific regulatory elements to obtain recombinant proteins specifically expressed in mammary gland. Among the variety of gene editing techniques, the CRISPR/Cas9 system is effective and specific in gene editing, showing a great advantages of gene knockout or knockin. This study was conducted to constuct a mammary gland bioreactor with specific expression of human insulin by using the CRISPR/Cas9 technology.In this study, the exon 2 of beta-casein gene locus was chosen as the exogenous gene insertion site, to construct the homologous targeting vector, the CNS2LA and CNS2RA of exon 2 of beta-casein was used as 5’and 3’homology region, hINS gene, negative screening DTA, bGH PolyA sequence as transcription termination signal were amplified by PCR, the mentioned elements were connected with the plasmid PⅢ-EGFP-PGK-Neo in the order, eventually a 9kb homologous targeting vector were constructed. And three sgRNA located in the exon 2 of β-casein gene were designed for construction of the CRISPR/Cas9 site-specific cleavage cutting vector. The two constructed vectors were confirmed by restriction fragment analysis and DNA sequencing, then they were co-transfected into the bovine fetal fibroblasts by liposome, transfected cells were screened by flow cytometry, and 6 strains of homologous targeting monoclonal cells were obtained after verified by PCR and DNA sequencing.To examined the hINS gene expression after transfection, the homologous targeting vector was transfected into the bovine fetal fibroblasts and mammary epithelial cells by liposome, and the cells after transfected were treated for 24 hours by (3-casein induced liquid, respectively, then the expression of hINS mRNA were detected by RT-PCR. The results show that hINS mRNA were not changed in fibroblasts with or without induced by β-casein induced liquid, but highly expressed in induced mammary gland cells, these results indicated that the P-casein promoter drive the expression of hINS in the homologous targeting vector. These results lays the foundation for the further cloning of transgenic cattles with specific expressing human insulin in mammary gland.