Preliminary Study On CRISPR/Cas9-mediated Site-specific Knockin Of IFN-? Gene In Goat Mammary Epithelial Cells

Mastitis is caused by infection with pathogenic microorganisms,and the damage caused to the dairy farming industry is very serious.Although the treatment of antibiotics can effectively curb the occurrence of mastitis,bacterial mutations and residues of antibiotics have seriously endangered the safety of humans and animals.Therefore,the use of biotechnology to produce animals with mastitis resistance is of great significance for the production of animals.Previous studies have shown that interferon(IFN-?)can regulate the body's immune system and inhibit the proliferation of bacteria that cause mastitis.In the pathogenesis of mastitis in dairy goats,IFN-? is an important anti-infective immune molecule,which can significantly reduce the infection caused by different bacteria.As a simple and efficient precise gene editing technology,CRISPR/Cas9 is widely used in the establishment of animal models and medicine.Therefore,the purpose of this study was to establish a mammary gland epithelial cell model of transgenic IFN-? gene by CRISPR/Cas9 system,which provides an important theoretical basis for the production of anti-mamming dairy goats transfected with IFN-?.The mammary gland tissue of the healthy Laoshan dairy goats in the lactation period was taken,and the goat mammary epithelial cells were cultured by digestion.The mixed fibroblasts were separated by several digestion passages.To determine the epithelial properties of the resulting cells,cells were identified by broad-spectrum keratin immunofluorescence staining.Human peripheral blood lymphocytes were isolated by kit,total RNA of lymphocytes was extracted by Trizol method,c DNA was synthesized by reverse transcription kit,and human IFN-? gene was amplified by RT-PCR.A knock-in site was designed for the goat casein gene.Design sg RNA on Cas-Designe(http://www.rgenome.net/cas-designer/)under OMICTOOLS,enter the sequence of the second exon and the eighth exon,respectively,and select 3 scores from each.High sg RNA was ligated into the lenti CRISPRv2 vector and sequenced to verify the correctness of the sg RNA sequence.By transfecting the correctly sequenced plasmid into goat fibroblasts,T7E1 digests the target sg RNA to target the goat genome,and selects a pair of sg RNA with higher targeting activity for subsequent experiments.The homologous recombination vector is transformed on the basis of the plasmid p CDH-CMV-MCS-EF1-cop GFP-T2A-Puro.The homologous recombination vector was constructed by restriction enzyme ligation.Design of homology arm: 5' homology arm is about 1000 bp upstream of goat CSN2 signal peptide(including signal peptide part);3'homology arm is partial sequence of eighth exon(including sg RNA,sg RNA is close to upstream),700 bp about.To initiate expression of exogenous IFN-? using the CSN2 promoter,the 5' homology arm and the IFN-? gene were seamlessly ligated together by overlap extension PCR.The constructed vector was sequenced to verify the correctness of the sequence of the ligated fragments.The 293 T cells were co-transfected with the packaging plasmid to express the lentivirus,and the mammary epithelial cells were transfected by lentivirus infection.The monoclonal cells were obtained by puromycin screening,and the cloned cells were identified by PCR.Identification primers spanning the homologous arm sequences were designed to identify whether homologous recombination occurred.The main findings of this experiment are as follows:(1)Goat mammary gland epithelial cells were obtained by digestion.(2)The CRISPR/Cas9 vector targeting goat casein was successfully constructed and identified by T7E1 enzyme.(3)The human IFN-? CDS region gene sequence was obtained by PCR amplification and sequencing,and the target(donor)vector p CDH-LA-IFN-cop GFP-Puro-SA transfected with human IFN-? gene was constructed,and in goat mammary gland The function of the targeting vector was verified in epithelial cells.(4)The goat mammary epithelial cell line transfected with human IFN-? gene was successfully constructed by CRISPR/Cas9 system mediated by the modified lentiviral vector.