Interference Effect Analysis Of RNAi Vectors Targeting Buffalo Inhibin Alpha Subunit Gene (INHA) And Construction Of Transgenic Mouse Model For The RNAi Vector

Buffalo is a very important draft animal in South China and has great potential in beef and dairy, but the reproductive function and lactation performance of buffalo are low; The inhibin (INH) can regulate FSH synthesis and secretion by negative feedback, which will reduce the reproductive performance of buffalo; Because of long gestation period and high cost of experiment of buffalo, we have to carry out RNAi effect research targeting buffalo INH by using experimental mouse model, which will provide a reference for the production of transgenic buffalo with high reproductive performance.In our study, we constructed RNAi vectors targeting INHA (inhibin a subunit) and analyzed the interference effect in the mouse ovary granulosa cells, through which we could get a better RNAi fragments with the highest interference effect among them. Then we transferred this RNAi fragment to the mouse genome using microinjection technique and studied the interference effect of the RNAi fragment in transgenic mouse in further. This will lay the foundation for the research of high fecundity transgenic buffalo.The main conclusions were as follows:(1) According to the buffalo INHA mRNA (EU884446.1) CDS, four shRNA were designed and they were:shRNA-b1, shRNA-b2, shRNA-b3 and shRNA-b4 (negative control).(2) Using PiggyBac transposon vector phi5 saved in our laboratory, we successfully constructed four RNA interference expression vector, namely. phi5siRNA-b1, phi5siRNA-b2, phi5siRNA-b3 and phi5siRNA-b4 (negative control).(3) Optimizing transfections using negative control vector phi5siRNA-b4. When usage amount of phi5siRNA-b4 was 750ng and the ratio of vector to lipofectamine was1:3, the highest transfection performance was acquired.(4) We transfected mouse ovarian granulosa cells with recombinant vector phi5siRNA-bl, phi5siRNA-b2, phi5siRNA-b3, phi5siRNA-b4 and another two RNAi vector phi5siRNA-3, phi5siRNA-4 saved in our lab. With qPCR technique, we got their interference efficiency, which were 47.9%,28.8%,30.7%,20.4% and 29.8%, respectively. Among them, phi5siRNA-bl had the highest interference performance (47.9%).(5) Phi5siRNA-bl was digested by SacI and ScaI and the fragment containing shRNA-bl and other expression element was purified by gel extraction, which would offer linear RNAi fragment for the production of transgenic mouse.(6) With the help of microinjection technique, purified RNAi fragment was microinjected into mouse zygotes (assisted by a company). We finaly got 60 newborn mice, among which 5(4♂ and 1♀) were identified as positive ones by conventional PCR.