Screening Of Shrna Sequences Of Buffalo Suv39H1Gene And Its Effect On Expression Efficiency Of Transgenic Cells

The expression of exogenous gene is influenced by many factors in transgenic animals. Suppressor of variegation3-9homolog1(SUV39H1) gene is a principal enzyme which is responsible for the accumulation of histone H3, containing dimethyl group or trimethyl group at its lysine9position (H3K9me2or H3K9me3), which plays a prominent role on expression efficiency of exogenous gene. The shRNA interference sequences targeting buffalo SUV39H1gene were screened, their effects on gene expression of exogenous IFNa-2b gene in Bcap-37cell line were examined, and the regulation mechanism was explored. It was looking forward to improving the expression efficiency of exogenous gene in transgenic animal.Construction of SUV39H1expression vector and screening of its shRNA fragments1.3’UTR and ORF sequences of buffalo SUV39H1gene were cloned, its miRNA sequence was predicted. The fusion expression vector of buffalo SUV39H1gene was constructed and its expression effect at the cellular level was detected. The results showed that, the homology of ORF and3? UTR sequences of SUV39H1gene between buffalo and cattle was99%and95%, respectively, the ORF sequence was more conservative than the31UTR sequence. The constructed recombinant plasmid of SUV39H1gene could express in293T cells. 2. The expression of SUV39H1gene in different stages of buffalo follicular development was detected by immunohistochemical technology. We found that the expression trend of SUV39H1gene in buffalo follicules was, accompanied by follicular maturation, its expression level gradually decreased. The expression level of SUV39H1was high in granulosa cells in all developmental stages of buffalo follicles, its expression in granulosa cells gradually increased accompanied by follicular maturation. In the oocyte of primordial follicles, expression of SUV39H1was strong both in the cytoplasm and the nucleus. Weak positive expression of SUV39H1gene was detected in the cytoplasm of buffalo cumulus oocyte complexes, negative expression was observed in the nucleus of all stage theca follicules.3.Based on the CDS sequence of buffalo SUV39H1gene, two shRNA fragments were designed and inserted into the lentiviral expression vector pSicoR-GFP, named as pSicoR-GFP-shSUV39H1/2and pSicoR-GFP-1864respectively. Confirmed by restriction enzyme digestion and sequencing, the shRNA plasmid and buffalo SUV39H1expression vector were co-transfected into293T cells at the ratio of3:1, the inhibition efficiency of shRNAs were detected by qRT-PCR and Western-blot analysis. The results of qRT-PCR showed that expression of SUV39H1gene in the negative and blank control group had no significant difference. Compared with the negative control group, the two shRNA fragments had significantly inhibition effect on expression of SUV39H1gene in the experimental groups (P<0.05), reached33.85%and78.75%respectively. Results of Western-blot analysis showed that expression of SUV39H1protein was clear in the negative control and blank groups, but decreased in two shRNA groups, consistent with the results of qRT-PCR.The effect of shRNAs of SUV39H1gene on expression of IFNa-2b and related genes in transgenic cells.High titer lentivirus containing shRNAs of SUV39H1was prepared by calcium phosphate transfection method. Bcap-37cells which steadily expressed IFNa-2b were infected by the virus, the cells were collected after72hours trasnfection and detected by qRT-PCR and Western-blot methods. The results revealed that shRNA1/2fragments of SUV39H1gene had obvious inhibition effect on the expression of SUV39H1gene at the same MOI virus infection condition, the inhibition efficiency were53.07%and31.28%respectively. Compared with the negative control group, the expression of IFNa-2b gene in transgenic cells infected with shRNAs virus was significant higher, increased by96.25%and121.08%respectively (P<0.05). In addition, compared with the control group, the expression of DNMT1、HDAC1and G9a gene reduced significantly, and the expression of the HAT1gene increased significantly (P<0.05). Regulation of SUV39H1gene would provide a way to improve expression of exogenous gene in transgenic animal.The above results revealed that the two shRNAs could effectively inhibit the expression of buffalo SUV39H1gene. The suppression of SUV39H1gene could improve the expression of IFNa-2b gene in Bcap-37cell line.