Lentiviral vector safety has been the impetus underlying the progress in packaging cell line development. The prospects of generating replication-competent lentiviruses (RCLs) and the potential for vector mobilization continue to be the driving force for the advancement of packaging cell lines. We have exploited the trans-lentiviral packaging system to develop the SODk3 packaging cell line for the generation of conditional self-inactivating (cSIN) vectors. Separating the gag-pol genome into two distinct expression cassettes (gag-pro and vpr-RT-IN) may reduce the potential for RCL formation, while concurrently employing cSIN vectors supports retention of the SIN phenotype in target cells and alleviates technical constraints associated with generating producer cell lines. Through development of the SODk3 packaging cell line we determined that the ratio of Gag/Pol in vector particles may be used as an indicator for packaging cell clones that yield high vector titers. Conditional SIN vector titers (1 x 10(7) TU/ml) were augmented through clonal selection. Distinct producer cell clones revealed a parallel between vector titer and transgene expression levels. We exploited this observation to demonstrate that incorporation of an internal ribosome entry site between the GFP marker and a relevant transgene affords efficient selection of high-titer producer cell lines. Furthermore, cSIN vectors generated from SODk3 packaging cells imparted efficient transduction of primary human fibroblasts, an indication of the future applicability of the SODk3 packaging cell line.