(M367) PRODUCTION OF A TELOMERASE-IMMORTALIZAED BOVINE SERTOLI CELL LINE.
Sartini, Becky1, Vogt, Volker1, Huang, Stephanie1, Parks, John1, 1 Cornell University, Ithaca, NY
ABSTRACT- Sertoli cells provide the architecture and metabolic support for germ cell differentiation in the seminiferous tubules of the mammalian testis. We report the production of an immortalized bovine Sertoli cell line by stable transduction of the cells with a retroviral vector encoding human telomerase reverse transcriptase (hTERT). Testis cells were isolated from a 19 week old bull by enzymatic digestion and Sertoli cells were enriched by differential plating and culture in vitro. First, the ability of in vitro-cultured Sertoli cells to be infected by a murine leukemia virus retrovirus vector was demonstrated with a vector expressing GFP. Then, fresh bovine Sertoli cell cultures were infected with vector expressing hTERT. The hTERT-infected cells were grown in culture for two months, representing 26 population doublings compared to 10 population doublings and eventual cell death in control-infected cell cultures. Expression of hTERT by infected cells was confirmed by RT-PCR and telomerase assay. Immunohistochemically, the hTERT-infected cells are positive for vimentin. Stem cell factor (SCF) and glial-derived neurotrophic factor (GDNF) gene expression was detected by RT-PCR in the hTERT positive cell population but not vasa, leukemia inhibitory factor (LIF), or p450scc expression. The hTERT-infected cells are a homogenous population of immortalized Sertoli cells and not germ, peritubular, or Leydig cells. This immortalized bovine Sertoli cell line is potentially useful as a feeder layer for germ cells in culture, for identifying specific factors critical to Sertoli-germ cell interaction, or for supporting the function of other somatic cells such as insulin production by cultured or transplanted pancreatic cells. This work was supported by USDA-NRI Grant 2001-35203-10836 and Genex Cooperative, Inc.
KEY WORDS: bovine, Sertoli, telomerase, retrovirus