p53 status and gene transfer experiments using CMV enhancer/promoter

Comparison of transfection efficiencies between different commercial reagen ts or methods is a matter of major concern in the held of gene therapy. Tra nsfection efficiencies are usually evaluated by the quantification of a rep orter gene expression (i.e., luciferase or lacZ) whose expression is usuall y driven by the CMV promoter. However, this experimental approach does not consider the possible effects of the transfection on the activity of the pr omoter used to drive reporter genes expression. Using p53 null fibroblasts we show that transfection efficiency estimated by the use of pCMV-luc or pC MV-beta gal plasmids may be dramatically affected by the cell p53 status. T hese data highlight the fact that differences in p53 levels may be one of t he parameters involved in the variation of transfection efficiencies observ ed with different cell lines. Furthermore, they point to the fact that comp arison of transfection efficiencies should distinguish differences in the e fficiency of transfection from differences in the level of transcription of the transgene. Finally they suggest that the known p53 down-regulation of the CMV promoter should be considered in order to avoid the nonintentional construction of transfer vectors in which the expression of a transgene dow n-modulates its own promoter. (C) 2001 Academic Press.