Pilot Study On Transduction And Transplantation Of Mouse Spermatogonial Stem Cells

Objective:By comparing the lipofection method with lentivirus-mediated transduction of mouse spermatogonial stem cells,to establish appropriate method of mice spermatogonial stem cells genetically modified in vitro,to obtain GFP-positive spermatogonial stem cells. Establish the platform of mice spermatogonial stem cell transplantation technology through the method of testicular efferent duct microscopy injection, in order to observe the survival situation of GFP-positive spermatogonial stem cells in the recipient mice testes.Method:Through the lentivirus, virus vector marked with the green fluorescent protein (GFP)gene is transducted into the spermatogonial stem cells which established before from newborn male ICR mice, and make it radiate Green fluorescent. Cultured and proliferated in vitro after collecting the GFP-positive cells, and then digested into cell suspension. Take an allotransplantation via efferent ductules of testis microinjection on the sterile mice which has been intraperitoneal injected with busulfan. Finally, observe whether or not the GFP-positive spermatogonial stem cell survival in the mouse testis and the process of spermatogenesis.Results:lentivirus transduction could be a efficient method to obtain GFP-positive spermatogonial stem cells.30 days after transplantation,the testis with scattered points of light could be seen under fluorescent dissecting microscope, however, no significant green fluorescent issued from seminiferous tubule, and a few of spermatogonial stem cells expressed GFP growing and proliferating along with the basement membrane of contorted seminiferous tubules in the testis frozen sections.Conclusions:1. It confirmed that Lentivirus-mediated is a suitable method for mice spermatogonial stem cell transduction.2. Established the mouse infertility receptor model by intraperitoneal injection of busulfan.3.Successfully completed the spermatogonial stem cell transplantation by using the efferent ductules of testis microinjection.4. Preliminarily investigated in the newborn ICR mice spermatogonial stem cell survival situation after cell transplantation.