Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis in male animals and have the potential value in producing transgenic animal by genetic modification and SSCs transplantation. However, SSCs from most mammalians are still difficult to be expanded in large scale and modified genetically in vitro. We recently succeeded in culturing mouse SSCs on a transgenic STO feeder layer expressing mouse glial cell line-derived neurotrophic factor stably. Here we report the successful in vitro transfection and transplantation of mouse SSCs based on our improved culture system. SSCs were purified using an anti-beta1 integrin antibody by panning method and differential plate, and cultured on transgenic STO feeder. Retroviral vectors PLNC-tPA and PL-tPA-SN carrying human tissue-type plasminogen activitor gene t-PA and the selectable marker neomycin phosphotransferase were constructed and introduced into packaging cell line PA317 by lipofectamine2000-mediated transfection. Five stable virus-producing cell clones, PA3171-5, were screened with the media containing G418. All the supernatants collected from these clones could infect standard tittering cell line NIH-3T3 and generate G418-resistant cell clone. Then supernatants containing the highest virus titer (1.6 x 105CFU/ml of PL-tPA-SN) were collected from PA3175 and used to transfect SSCs maintained on transgenic STO feeder. Subsequently, the transfected cells were microinjected into the seminiferous tubules of 30 recipients intraperitoneal injected with 40 mg/ml busulfan one month ago. 35 days later, both the destroyed seminiferous tubules and the decreased testis weights of 22 recipients (73.33%) recovered significantly (p<0.05) compared with controlled groups. PCR of testis DNA and sperm DNA also indicated the presence of human tPA gene in these recipients. The present results suggest that mammalian SSCs can be propagated and genetically modified by using an appropriate culture system. It is also implied that allogeneic SSCs transplantation is possible between nonimmunosuppressed, histoincompatible males. This research was supported by National Natural Science Foundation of China (Grant No. 30200195, 30471246, 30771555). (poster)