Introduction of a selectable gene into murine T-lymphoblasts by a retroviral vector

A recombinant retroviral vector with an inserted bacterial neomycin resistance (neo) gene was used to transfer in vitro neomycin resistance (neoR) into murine cytotoxic lymphocyte precursors (CLP). The infection protocol involved co-cultivation of mitogen-activated splenic T-blasts with irradiated cells that produced either the recombinant retrovirus plus a helper virus, or exclusively the recombinant retrovirus. Infected T-blasts were subsequently cultured under limiting dilution (LD) conditions that supported clonal in vitro development of a large fraction of murine CLP. In infected T-blast populations, frequency estimates were obtained for CLP that developed into functional cytotoxic T-lymphocyte (CTL) populations under G418-selected or non-selected conditions; from these frequency estimates, an efficiency of transduction of the neoR phenotype into murine CLP of 2-8% was calculated. Some conditions were defined that influenced transduction efficiency, i.e., the density of the infecting monolayer cells; the presence of interleukin 2-containing conditioned medium and mitogenic lectins during the co-culture period; a delayed onset of G418 selection after infection. It was demonstrated that the neoR phenotype of functional CTL populations derived from infected CLP resulted from expressed recombinant retrovirus.