USE OF BICISTRONIC RETROVIRAL VECTORS ENCODING THE LACZ GENE TOGETHERWITH A GENE OF INTEREST - A METHOD TO SELECT PRODUCER CELLS AND FOLLOW TRANSDUCED TARGET-CELLS

The coordinate expression of a marker gene and a therapeutic gene in o ne retroviral vector has considerable advantages. High-titer producer lines can potentially be selected on the basis of marker gene expressi on, and the expression of transduced genes in target cells can readily be followed. Moreover, target cells with stable high expression can b e selected before use in therapeutic protocols or research questions. We used internal ribosomal entry site (IRES) sequences to express two genes in the same retroviral vector. We used the LacZ gene as the mark er gene and the cytokine interleukin (IL)-7 or dominant negative (dn) forms of the T-cell tyrosine kinases ZAP-70 and lck as genes of intere st. Amphotropic packaging cells transfected with MFG-IL-7-IRES-LacZ, M FG-dnZAP-70-IRES-LacZ, or MFG-dnlck-IRES-LacZ were sorted on the basis of beta-galactosidase expression. These LacZ-positive producer cells also expressed the gene of interest, produced high-titer retrovirus, a nd were capable of efficiently transducing jurkat T cells and T-cell c lones. When MFG-IL-7-IRES-LacZ-transduced jurkat T cells were sorted o n the basis of LacZ expression, a positive correlation with the amount of IL-7 produced by these cells was found. This demonstrates that sel ection of the LacZ marker gene also selects for cells that express the gene of interest at high levels. Moreover, T cells transduced with th e dn tyrosine kinases and selected on the basis of LacZ expression sho wed functional alterations after T-cell receptor stimulation, demonstr ating that retrovirally transduced signaling molecules can alter the f unction of T cells.