Expression And Identification Of Egfp Expression Vector Containing OSP-1 Promoter And Two Promoters CMV-OSP1

Reproductive rate is an important economic trait in goat reproduction. To improve the fecundity, also known as litter size, is of special meaning in the selection of animals with high reproductivity. Ovary is the main functional organ in reproduction, and Granulosa cells playe an important role in maintaining the function of dairy goat breeding.It is a hot topic in animal reproductive science to screen and research ovarian tissue specific promoter, which could regulate the genes related with lambing performance in the ovarian granulosa cells.Currently, it was found that the ovarian tissue-specific promoter were only OSP-1, OSP-2. But there lack experimental proof whether these promoter can regulate functional genes associated with the litter size-specific in the dairy goat ovarian tissue.This study was designed to analyse the ovarian tissue-specific of OSP-1 and the expression efficiency of EGFP in the ovarian tissue with the regulation of two promoters, with the use of the constrction of ovarian specific promoter expression vector, the isolation and cultivation of granulosa cells, lipofection, real-time quantitative PCR and so on. In this research, we design and clone ovarian-specific promoter1 (OSP-1). Three vectors carried different promoters were constructed, which were carrying EGFP driven by CMV promoter, OSP-1 promoter and CMV-SP conjoint promoters, respectively. Those were named: pCMV-EGFP, pOSP1-EGFP and p CMV-OSP1-EGFP.In this study, the use of mechanical separation have successfully isolated and cultured ovarian mural granulose cells of dairy goat, and most of primary adherent granulose cells were spindle or irregular polygon. After the inoculation, cells were suspended spherical shape; with the increase of culture time, cells begin to contact and adherence, while there are close to each movement and the emergence of the phenomenon of cell clusters. Continuing to been cultured, the pseudopods in the bottom of cell clusters contact with each other. After 5-6 days, the bottom of petri dish can be covered, and form a granular cell layer.The vector pOSP1-EGFP have been transfected into four different cell: ovarian mural granulose cells of dairy goat, breast epithelial cells, fetal fibroblast cells and human ovarian cancer cell line HO-8910. The expression of OSP-1 were been detected only in the ovarian mural granulose cells of dairy goat and HO-8910, after observed by inverted fluorescence microscope and real-time quantitative PCR. This shows pOSP1-EGFP has the specific expressional activity in ovarian tissues.The EGFP expression vectors containing OSP-1, CMV and CMV-OSP1 promoter have been transfected into different cell. We detected the level of EGFP expression of different promoters in different cells by real-time quantitative PCR. The results showed: in the ovarian mural granulose cells of dairy goat, the expression efficacy of EGFP by two promoters was higher than the groups of pOSP1-EGFP and pCMV-EGFP (29%, P<0.01 and 51.22%, P<0.01); in the human ovarian cancer cell line HO-8910, the expression efficacy of EGFP by two promoters was higher than the groups of pOSP1-EGFP and pCMV-EGFP (21%, P<0.01 and 44.07%, P<0.01). These data indicated that it can improve the efficiency of exogenous gene expression effectively.