Effects of infection rate and selection pressure on gene expression from an internal promoter of a double gene retroviral vector.
Many commonly used retroviral vectors express one gene from the viral long terminal repeat (LTR) promoter and another gene from an internal promoter. We have investigated factors affecting the expression of the luciferase reporter gene from the internal cytomegalovirus-derived promoter of the retroviral vector, LNCX, which contains a LTR-driven neo gene as a selectable marker. A subline of human HT1080 cells, expressing the murine ecotropic receptor, was infected with retrovirus generated by transient transfection of BOSC 23 packaging cells. Mass populations of cells infected under conditions resulting in different initial infection rates (IIR) and selected with G418, showed highly variable luciferase activity. Luciferase expression in cell populations with IIR < or = 5% was generally low; many populations with IIR < 1% had marginal or no luciferase activity. The loss of luciferase expression in low-IIR populations was associated with G418 selection. In contrast, cell populations with IIR > or = 6% showed higher luciferase expression, which was strongly correlated with the IIR. Southern hybridization analysis showed that most cells of the low-IIR populations carried one integrated provirus, with a high incidence of structural rearrangements that abolished luciferase activity. In contrast, populations with IIR > or = 6% contained two or more copies of integrated provirus per cell, and their luciferase activity correlated with the provirus copy number. Luciferase expression was relatively stable in the populations with IIR > 1% maintained in the absence of G418. Increasing the selective concentration of G418 or prolonged maintenance of cell populations in the presence of G418 resulted in higher incidence of provirus rearrangements and decreased luciferase expression. These results indicate that the negative effect of selection for the LTR-driven gene on gene expression from an internal promoter depends on the selection stringency and can be obviated by increasing the infection rate.
Schott
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B
Department of Genetics, University of Illinois at Chicago 60607-7170, USA.
Iraj
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Roninson
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D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D011401:Promoter Regions, Genetic; D011533:Proviruses / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic; D014162:Transfection
D004279:DNA, Viral; D008156:Luciferases