Quantitation of Retroviral-Mediated Transfer Using Luciferase in Living and Lysed Cells

Abstract

We have developed a murine retroviral vector containing an improved luciferase gene for the study of retroviral gene transfer and expression in living or lysed cells. We used a cytosolic form of luciferase gene (luc+) with transcriptional enhancements that yielded greater expression levels. The luc+ gene was subcloned into the retroviral plasmids pDON-AI, in which almost the entire U3 region has been replaced with the heterologous human cytomegalovirus immediate-early promoter. A stable ecotropic and amphotropic retrovirus-producing cell line was generated with a titer 1 × 106 cfu/mL. NIH/3T3(tk-) cells transduced with ecotropic luciferase retrovirus demonstrated a high level of luminescence on the third day. Lysed NIH/3T3(tk-) cells demonstrated a 10-fold increase in activity as compared to living cultures. The creation of a new retroviral system allowed a substantial decrease to 5 days from the 10–14 days previously needed to evaluate viral transfer using the standard neomycin method. Our assay also provides a quantitative assessment in contrast to the β-galactosidase detection method, which also takes 5–6 days but lacks quantitative evaluation. Thus, the expression of an integrated luc+ gene in eukaryotic cells provides a powerful tool for the study of retroviral gene transfer and will greatly facilitate functional studies in both living and lysed cells.