Figure 6

Heterologous MLV RNAs form, predominantly, 1-LTR episomal cDNAs following delivery with HIV-1 viral particles. MLV/HIV RRE + RU5PS chimeric vector was packaged into HIV-1 and MLV viral particles in the presence of Rev. 293T cells were transduced with equivalent transducing units for HIV-1 and MLV packaged vectors. Total cellular DNA was harvested at 5 days post-transduction (episomal and integrated vector DNA, P0), and after five cell passages (integrated vector DNA, P5). Vector DNA copy number as measured by qPCR to the WPRE A, and β-globin DNA copy number B, were determined by qPCR. Data are shown without cell passages (P0; white bars) and after 5 cell passages (P5; black bars). Vector DNA copy number was normalized to β-globin copy number C, and fold decreases in vector DNA levels, after passaging cells, are shown. Error for all bar graphs is expressed as ±S.D. D. Southern blot analysis of total DNA isolated from 293T cells transduced with MLV/HIV RRE + RU5PS vector packaged into either HIV, or MLV, viral particles (as indicated above lanes). Total DNA was isolated at 5 days posttransduction (P0) and after 5 passages of cells (P5). DNA was digested to distinguish between 2-LTR, 1-LTR, and linear episomal forms, as well as the vector backbone which is indicative of integrated vector DNA after passaging cells.