Exploration of critical parameters for transient retrovirus production.

The pCL system was developed to aid in the production of retrovirus that encodes cytotoxic or cytostatic cDNA's. A principal feature of this system is the transient production of virus after co-transfection of the viral and packaging vectors in the 293T cell line. This approach obviates the need for selection of the producer cells, thus minimizing potential affects of the encoded genes. However, the transient nature of this system also creates a number of experimental variables. In this study we have examined and optimized elements related to the production of the pCL retrovirus. For example, co-transfection of the packaging sequence along with the viral vector has been optimized in terms of both the total amount of DNA transfected and the relative proportion of each plasmid. We have also tested the affect of increased synthesis of viral proteins in the producer cells and the kinetics of virus accumulation in the supernatant. These findings may be of interest to those who use pCL or any transient packaging system in their gene transfer studies. In addition, these studies may aid in the validation and development of transient retrovirus production systems for clinical applications.