A retroviral vector that allows efficient co-expression of two genes and the versatility of alternate selection markers.

Abstract

pZIG(hGCSFR) is a retroviral vector that can co-express two genes and also provides alternative selection markers. This retroviral vector has been constructed to incorporate an internal ribosome entry site (IRES) element to co-express two exogenous genes in mammalian cells. Two marker/selection genes have been cloned into the vector that not only allow the efficiency of co-expression to be quantified but also provide versatility of selection criteria. A cDNA encoding the hGCSFR (signaling deficient) was cloned as the 5' cistron, and a gene encoding a neomycin-resistance and beta-galactosidase (beta geo) fusion protein was cloned as the 3' cistron. The two marker genes cloned in this retroviral vector allow the selection and isolation of mammalian cells following either transient (by fluorescence-activating cell sorting analysis of hGCSFR-positive cells) or stable (neomycin resistance) infection with the certainty that over 93% of clones will co-express both genes. This novel vector offers a versatile retroviral vector that can be potentially used in a wide range of gene therapy applications as the gene of interest can be coexpressed with either of the marker genes depending on whether the retroviral infection is to be performed in vitro, in vivo, or ex vivo.