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Quantitation of retroviral-mediated transfer using a luciferase gene Vassili A Zakhartchenko*1; W. French Anderson2; Yanina Rozenberg3 (1) CCMB, University of Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033, USA; (2) Gene Therapy Labs, University of Southern California, Norris Cancer Center, 1441 Eastlake Avenue, Room 6316, Los Angeles, CA 90033, USA; (3) Gene Therapy Labs, University of Southern California, Doheny Vision Research Center, 1355 San Pablo Street, Room 205, Los Angeles, CA 90033, USA      *(zakhartc@hsc.usc.edu) We designed a murine retroviral vector containing a luciferase gene to study retroviral gene transfer and expression. We used the newly developed cytosolic form of luciferase gene (luc+) with several modifications of the transcriptional region yielding greater expression. The luc+ gene was cloned into retroviral plasmid pDON-AI in which almost the entire U3 region was replaced with the heterologous human cytomegalovirus immediate-early promoter. Stable ecotropic and amphotropic retrovirus-producing cell lines were generated. NIH/3T3 cells transduced with ecotropic luciferase retrovirus demonstrated a light output 24 hours after transduction as tested in living cells. The sensitivity increased 100 times when the luciferase assay was done by lysing the cells. The use of retrovirus containing luciferase gene significantly reduces the time of titer determination. In comparison with neomycin method, which usually takes 10-14 days, the novel retrovirus construct allows analysis of reporter expression 24 hours after transduction. Thus, the expression of an integrated luc+ gene in eukaryotic cells provides a powerful tool to study retroviral gene transfer, expression and functional studies. [Poster: zakhartc.vassil.27632]

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