Internal Ribosomal Entry Site-Containing Retroviral Vectors with Green Fluorescent Protein and Drug Resistance Markers

Abstract

To facilitate gene delivery into animal cells we developed and characterized a family of single-transcript vectors (STVs) with different selection markers expressed from the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV). Retroviral IRES-STVs (R-IRES-STVs) were assembled using an LNCX backbone (Miller and Rosman, 1989). In all of these constructs, a multiple cloning site (MCS) is located immediately downstream of the single promoter and is followed by the IRES sequence and a selectable marker. This configuration ensures that the MCS-inserted gene will be expressed in selected cells. The selectable markers of these vectors provide resistance to G418, puromycin, hygromycin B, histidinol D, and phleomycin. One STV contains green fluorescent protein (GFP) as a selectable marker, permitting FACS-mediated selection, which may prove useful in gene therapy applications. More than 70% of recipient cells could be infected with R-IRES-STVs after one round of infection. Up to 99% of infected cells expressed the reporter gene (GFP) after selection with an appropriate drug. When ecotropic receptor was delivered via R-IRES-STV into human HT1080 cells, populations of drug-selected cells as well as a majority of individual clones were found to be highly susceptible to infection by ecotropic retroviruses.