Establishment and identification of immortalized sheep ovarian granulosa cells

Background Follicular granulosa cells are an important cell source for studying animal reproductive function. Results The aim of this study was to investigate the gene transfection of sheep ovarian granulosa cells (GC) mediated by SV40 T lentiviral vector. The effects of GC immortalization and functional properties were verified. The results showed that the cell line could be continuously passaged to the 50th generation. And the cell morphological characteristics and fluorescence expression intensity remained stable. By optimizing the infection conditions (MOI = 10), efficient transfection of GFP-tagged SV40T gene was successfully achieved (efficiency of about 90%)(P<0.05). The immortalized sheep granulosa cell line (GCs-SV40T-GFP) was successfully established. The growth rate of immortalized cells was significantly higher than that of primary GCs-1 cells. Flow cytometry showed that the proportion of S phase and G1 phase of immortalized cells increased significantly (P < 0.05). WB and q-pcr confirmed that the transfected cells continued to secrete estradiol to maintain endocrine function (P < 0.05). In addition, in vivo safety evaluation showed that GCs-SV40T-GFP cells did not induce mouse tumors. Conclusions In this study, a functionally stable immortalized sheep granulosa cell model was successfully constructed. At the same time, it also provides an important tool for animal reproductive function research and hormone regulation mechanism exploration.