p53 Status and gene transfer experiments using CMV enhancer/promoter.
Comparison of transfection efficiencies between different commercial reagents or methods is a matter of major concern in the field of gene therapy. Transfection efficiencies are usually evaluated by the quantification of a reporter gene expression (i.e., luciferase or lacZ) whose expression is usually driven by the CMV promoter. However, this experimental approach does not consider the possible effects of the transfection on the activity of the promoter used to drive reporter genes expression. Using p53 null fibroblasts we show that transfection efficiency estimated by the use of pCMV-luc or pCMV-betagal plasmids may be dramatically affected by the cell p53 status. These data highlight the fact that differences in p53 levels may be one of the parameters involved in the variation of transfection efficiencies observed with different cell lines. Furthermore, they point to the fact that comparison of transfection efficiencies should distinguish differences in the efficiency 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 down-modulates its own promoter.
Allamane
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INSERM U318, CHU Michallon, Grenoble cedex 09, 38043, France.
Ratel
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Jourdes
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Berger
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Benabid
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Wion
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D000818:Animals; D003587:Cytomegalovirus / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D017930:Genes, Reporter; D016158:Genes, p53; D008156:Luciferases / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D011401:Promoter Regions, Genetic; D014162:Transfection / Q000379:methods; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency / Q000502:physiology*; D001616:beta-Galactosidase / Q000235:genetics
D016159:Tumor Suppressor Protein p53; D008156:Luciferases; D001616:beta-Galactosidase