Clone And Eukaryotic Expression Rad51 Gene In Ovis Aries

Gene of Rad51 can lead the homologous recombination repair, help to maintain the stability of gene, resist cytotoxicity factor harm to DNA. In this study, by construct expression carriers of Rad51, which comes from sheep, at the same time, express the Rad51 protein in fibroblasts, as well as contrast Rad51 gene expression level after transfect different plasmids which act on same genetic fragments. To discuss the efficiency of homologous recombination and raising the number of positive monoclonal cells and laid the theoretical and experimental basis for ovis aries Rad51 gene cloning is our purpose.Methods:In experiment 1, got the ear tissues from suffolk ewe, aged 18 months old. To get high purity of fibroblasts of sheep, by the steps of digested the tissues, primary culture ovis aries fibroblast, passage culture, frozen storage and resuscitation of cells in two ways(IV Collagenases and Tryp LETM Select). In experiment 2, designed the c DNA PCR primers of Rad51, the production inserted to pc DNA3.1(+), which has Afl II and Xho I restriction enzyme cutting site, constructed the eukaryon expression vector pc DNA3.1-Rad51, enzyme digestion after PCR Rad51 sequence, then sequencing the expression vector. In experiment 3, transfected 4μg Rad51 expression vector, CRISPR/Cas9 Plasmid and TALENs plasmids into fibroblasts(2×106) with transfer Buffer(100μL) and program of CZ-167 partly. To detect the plasmid expression in the cells, made the p MAX plasmid as negative control. In experiment 4, quantitative real-time PCR and western blot examination detect the Rad51 expression level of fibroblast in m RNA and protein level expression, experimental datum analyzes with the LSD statistical method.Results:Experiment 1 showed that: Got high purity fibroblast cells of ovis aries by two steps enzymatic digestion and differential adhesion, which were with good growth state, generally 2 days could be full of 60 mm cells dish after adherent. Experiment 2 showed that: Rad51 gene c DNA fragments could obtain via PCR ovis aries genome. Ovis aries Rad51 sequence contrasts with NCBI announcement sequence, we found that start from the initiation codon(ATG), the 90 th basic group changed A to G(NCBI is G). Amino acid analysis showed that although 90 th basic group changed, that have not changed the amino acid, the rest of sequences are completely consistent. Experiment 3 showed that: fibroblasts infection p MAX plasmid about 72 h could be expression green fluorescent protein, cells in the 6 well plate convergence degree could reach more than 80%. Experiment 4 showed that: DNA double strand break, the Rad51 gene significantly is higher than the normal cell in the m RNA level, the protein expression extremely significant difference compared with normal cells, different ways of DNA rupture, Rad51 gene expression quantity also is not the same.Conclusion : Comprehensive analysis showed that: pc DNA3.1(+) as the framework inserted c DNA could be constructed ovis aries Rad51 expression vector. Electric transfection of CRISPR/Cas9, TALENs, pc DNA3.1-Rad51, p MAX plasmid into fibroblast foud that Rad51 m RNA and protein expression was significantly higher than that in normal fibroblasts.