Construction Of Double RNAi And Ovarian Specific Expression Vectors Mediated By PB Transposon And Efficiency Analysis

Water buffalo, is an important species in South China, as the characteristics of disease resistance and superior milk with high quality. However, the lower reproduction performance has directly restricted the development of water buffalo industry. Inhibin (INH) and follistatin (FST) are the local factors of folliculogenesis via regulating the release of follicle-stimulating hormone (FSH) and Luteinzing hormone (LH) in an autocrine and paracrine manner in different species. Based on these functions, researches about them have been studied in many domesitc species but less in water buffalo, therefore, we think it is necessary to develop the breeding techniques of water buffalo from this aspect. As a DNA transposon, PB transposon has become a powerful genetic manipulation for generating transgenic animals and insertional mutagenesis. In this study, PB transposon-derived double RNAi vector was established and the efficiency was analyzed at the cellular level to select the transgenic cell line. In addition, we also established two ovarian specific expression vectors and tested their specific expression and validity. All of these would strengthen the basis on the mechanism of folliculogenesis and the techniques of transgenic buffalo.The main results are as follows:(1) We designed the shRNAs of which the target gene are INHA and FST, and constructed the double RNAi vector pB-H1-shRNA1-U6-shRNA2which contains two expression cassttes;(2)The BuGCs and BGCs were transfected by the double RNAi vector, and the BuGCs and BuFFs were co-transfected by the transposase and double RNAi vector. By compairing the different cells with different transfection, we found that cells co-transfected integrated forigen gene stably, while the cells transfected solely only for transient expression. In addition, the efficiency of co-transfection is higher than the sole one.(3)We investigated their inhibition to the mRNA of INHA and FST in BuGCs and BGCs. The highest activity was displayed in INHA shRNA in co-transfected BuGCs, of which the inhibition rate reached37.37%. At the same time, the inhibition rate of FST shRNA in solely-transfected BGCs was the lowest (only3.55%); (4)After using G418for co-transfected cells, we observed the level of GFP constantly in cells. After7days, BuGCs and BuFFs by co-transfected both expressed GFP stably. Besides, the fragment of PB transposon was amplicated by PCR with the transfected cells’DNA. The transgenic cell line was producted and presented stable inhibition.(5) We constructed two expression vectors based on PB transposon, which contained two expression promoter CYP19and OSP-1(donated from Guangxi University), respectively.(6) FSHβ was amplificated and inserted into downstream of promoters to construct two FSHβ expression vecotors.(7)The two expression vectors were transfected to BuGCs and BuFFs, respectively. We found the expression of FSHβ in BuGCs but not in BuFFs via Q-PCR. It confirmed that the expression vector only can translate with BuGCs in ovarian to express FSHβ, which proved the CYP19and OSP-1promoters were all ovarian specific promoters. However, the expression validity of OSP-1promoter was significantly higher than that of CYP19promoter. Therefore, the CYP19promoter could be considered as a potentially expressed promoter in ovary.