Gene Transfection Of Spermatogonial Stem Cell Proliferation And Differentiation In The Receptor Microenvironment

The spemratogonial stem cell (SSC) is the fundation of spermatogenesis and it continues to divide throughout the lieftime of a male. It has abilities to self-renew and differentiate into spermatozoa. Spermatogonial stem cells are the only stem cells that are capable of transmitting genetic information to subsequent generations in adult animals. Therefore,SSCs are not only the study object of stem cell biology,but also the valuable resource of in vitro spermatogenesis,gene analysis and functional genomics.Spermatogonial stem cell tnansplantation(SSCT) means that donor testis cells can be transplanted to recipient testes where they re-establish spermatogenesis. SSCT was first reported by Ralph Brinster's laboratory in 1994. It has proven to be a technological breakthrough in the study of both stem cells and Sertoli cell-germ cell interactions. After SSCT ,some correlative techniques ,such as the enrichment and long-term culture of stem cell populations and cryopreservation etc, were developed quickly. It is a new way for studying the spermatogenesis and producing the transgene animals to combine SSCT with the genetically modified SSCs. It provides abroad avenue for academic research and practical application in basic seienee, human medicine,and domestic and wild animal reproduction. Because of those advantages of SSCs and SSCT, especially in the transgene animal field,our study will focus on the method of genes transferred into SSCs, the self-renewing and differentiating of the doner cells in recipient testes after transplantation.In this study, we extracted pmaxGFP plasmid. Then SSCs were isolated and enriched by using two-step enzymatic digestion according to provirously described and the adherence selection .Green Fluorescent Protein(GFP) genes were mediated by FuGENE HD Lipidsome to spemratogonial stem cells of donor mice in vitro. After that,these modified SSCs were transplanted into the seminiferous tubules of anesthetized recipients depleted of germ cells by busulfan treatment. The testes were excised in different time after transplantation. We observed the testes under fluorescence microscope and used the paraffin-cut sections and PCR tools respectively to analyse and explore the behavior of donors spermartogonial stem cells with exogenous gene in recipient testes after transplantation.The result showed: Firstly, the success of transplantation with spermatogonia demonstrated that our technique for the cell transplantation was reliable in the research. And 3 months after the transplant operation, spermatogenesis of the donor stem cells could be observed clearly and the male have reproduced progeny after mating with normal female. Secondly, the efficiency of genes transfer mediated by FuGENE HD Lipidsome which was used in our study is about 10%. However,we haven't obtain the mice expressing GFP yet. So we don't know whether it is the immediate tranfer now. And because the time is limited, we should keep on observing those transgene animals.In our study,the efficiency of transfer is not high. There are some reasons for this problem. First, the population of SSCs is small in the testis.Second,SSCs are not cultured continuously like embryonic stem cell.Third,the cycle of the SSCs' self-renew is long.However,we can foresee that producing transgene animal using SSCs is a potential mathod once we overcome these details of technique.