Study On CRISPR/Cas9 Mediated Knock- In Of Human TNF-α Gene In Bovine Fetal Fibroblast

Animal mammary gland bioreactor is a transgenic technology for development of recombinant proteins, in which the foreign gene is expressed under control of mammary gland-specific regulatory elements. CRISPR/Cas9 technology has characters of high targeting efficacy and a robust of sequence-specificity. It has a broad range of applications value in introducing gene knock-out and knock-in in the specific sites. Tumor necrosis factor-α (TNF-α) is a kind of important cytokine, is mainly secreted by activated macrophages and monocytes, has roles of tumor lethal activity, immune regulation, inflammation promotion and antiviral enhancement and so on. In this study, the homologous recombination vector with human TNF-a gene and a CRISPR/Cas9 vector targeting to the second exon of beta-casein gene locus were constructed. Then, the two vectors were co-transfected into the bovine fetal fibroblasts by electroporation. After screened with G418, positive clone cells were examined for the interation site by PCR. The results indicate that the exogenous gene was inserted correctly into the specific site of β-casein. To test the specifically expression of the exogenous gene TNF-α mRNA in mammary gland, the homologous recombination vector and the CRISPR/Cas9 vector were co-transfected into the bovine mammary epithelial cells, RT-PCR and Western Blot were used to detect the expression of mRNA and protein of TNF-α, results showed that bovine mammary epithelial cells expressed mRNA and protein of TNF-α gene. This study is of great importance for further study of producing of TNF-a transgenic cow.