Tumor-specific genes delivered to dendritic cells (DCs) have been used for the generation of cytotoxic T cells (CTLs), but their application has been limited on the one hand by low viral titers resulting in low transduction efficiency and poor protein production, and on the other hand by immunogenicity of the selectable marker and poor viability of the DCs. We addressed these limitations by creating a multipurpose master vector (pMV) and cloning the tumor gene NY-ESO-1, which is highly expressed in more than 50% of advanced myeloma patients. pMV was constructed from a Moloney murine leukemia virus (Mo-MuLV)-based retroviral backbone with the following features: (1) an extended packaging signal to achieve high viral titers, (2) a splice acceptor region to facilitate protein production, (3) a nonimmunogenic selectable marker, dihydrofolate reductase-L22Y (DHFR(L22Y)), to exclude the generation of CTLs against the selectable marker, (4) an internal ribosomal entry site between the tumor-specific gene (NY-ESO-1) and the selectable marker DHFR(L22Y) for coexpression of two heterologous gene products from a single bicistronic mRNA, minimizing the possibility of differential expression of these two genes, and (5) human granulocyte-macrophage colony-stimulating factor (hGM-CSF) cDNA driven by the human T-lymphotropic virus promoter to enhance DC function and viability. Recombinant virus of pMV-NY-ESO-1 was generated with vesicular stomatitis virus G envelope protein (VSV-G) in the GP2-293 cell line for efficient transduction. We present evidence that the DC phenotype is unaltered after transduction and that more than 85% of DCs express NY-ESO-1, which secrete approximately 40 ng of GM-CSF per 10(6) DCs.