Study On Culturing Spermatonial Stem Cells Of Mice Fed With NIH-3T3 Cells Of Trangene GDNF

Spermatogonial stem cells (SSCs) have the abilities to self-renew and differentiate into sperm, are the only type of stem cells that can transmit genetic information to the next generation, and the initial cells that maintain spermatogenesis. It is known that the spermatogenesis is a complex and highly organized process, and the mechanism controlling spermatogenesis was largely unknown. Interestingly, the proliferation and induced differentiation of spermatogonial stem cells would provide a new approach for the study and elucidation of spermatogenesis. Moreover, the in vitro culture of spermatogonial stem cells in combination with transplantation technique will make possible the genetic therapy of human genetic diseases, or make transgenetic animals via in vitro manipulation of SSCs. Importantly, the in vitro culture of SSCs, combined with other biological technologies, have shed light on the protection of endangered anmals, the injury recovery of endogenerous SSCs after chemotherapy and irradiation therapy, as well as restoration of male fertility.However, the viability and proliferation of SSCs were affected by many factors including the culture medium, additives added to the medium, the concentration of serum, growth factors, hormones and vitamins. In addition, the culture systems of SSCs with different genetic origin have been shown to be different at a large degree. Also, it was reported that serum and testis somatic cells induce the differentiation of SSCs in vitro. Therefore, the enriched SSCs and serum-free defined culture medium are crucial for the maintenance of SSCs in vitro. It has recently reported that glial cell line-derived neurotrophic factor (GDNF) or its signaling pathways was the central factor and signaling pathways of SSC self-renewal.In the present study, we extracted the total RNA form mouse testis tissue, designed primers of GDNF gene dependent upon the known sequence of GDNF cDNA, and cloned the GDNF gene by RT-PCR. We then structured a plasmid vector containing mouse GDNF gene, and named it pcDNA3.1-GDNF. The NIH-3T3 cells were transfected with this vector by lipofectamine 2000, sorted positive cell colonies via G418 selection. Results of RT-PCR, immunocytochemistry and western-blot indicated that we gained the cell line with stable expression of exogenous GDNF.