Quantitation of retroviral-mediated transfer using luciferase in living and lysed cells.
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 x 10(6) 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 beta-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.
Zakhartchenko
V A
VA
Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles 90033, USA. zakhartc@hsc.usc.edu
Anderson
W F
WF
Rozenberg
Y
Y
D016475:3T3 Cells; D000818:Animals; D003587:Cytomegalovirus / Q000235:genetics; D003600:Cytosol / Q000201:enzymology; D015870:Gene Expression; D017781:Genes, Immediate-Early; D005822:Genetic Vectors; D008156:Luciferases / Q000235:genetics*; D008163:Luminescent Measurements; D051379:Mice; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics*; D013997:Time Factors; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D008156:Luciferases; D001616:beta-Galactosidase