Transfection techniques affecting Stat3 activity levels.
Transfection techniques, such as calcium-phosphate or liposome-mediated gene transfer, are commonly used for the examination of the effect of a gene upon cellular phenotype and biochemical properties. We previously demonstrated that cell to cell adhesion causes a dramatic increase in Stat3 activity. Given that the opportunities for cell to cell adhesion could be altered due to the presence of the DNA-containing complexes, we examined the effect of the calcium-phosphate transfection procedure upon Stat3 activity levels. The results revealed a dramatic increase in Stat3 phosphorylation at the critical tyr705 site and Stat3 activity following calcium-phosphate transfection. This increase was noted even in the absence of DNA and was not due to the mere presence of calcium ions. In contrast, DNA introduction through electroporation or infection with a retroviral vector did not affect Stat3 activity, while cationic lipids such as lipofectamine or Fugene6 had a less pronounced effect than calcium-phosphate transfection. These results indicate that caution is required in the interpretation of results with regard to activity of Stat3 following certain commonly used transient transfection regimens.
Arulanandam
R
R
Department of Microbiology and Immunology and Department of Pathology and Molecular Medicine and Cancer Research Center, Queen's University, Kingston, Ont., Canada K7L 3N6.
Vultur
A
A
Raptis
L
L
D000818:Animals; D002130:Calcium Phosphates; D004268:DNA-Binding Proteins / Q000502:physiology*; D018274:Electroporation; D009052:Leukemia Virus, Murine / Q000235:genetics; D007942:Leukemia, Experimental / Q000503:physiopathology; D008055:Lipids; D008081:Liposomes; D051379:Mice; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D012192:Retroviridae Infections / Q000503:physiopathology; D050796:STAT3 Transcription Factor; D015398:Signal Transduction / Q000502:physiology; D015534:Trans-Activators / Q000502:physiology*; D014162:Transfection / Q000379:methods*; D014412:Tumor Virus Infections / Q000503:physiopathology
D002130:Calcium Phosphates; D004268:DNA-Binding Proteins; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D050796:STAT3 Transcription Factor; C494087:Stat3 protein, mouse; D015534:Trans-Activators; C020243:calcium phosphate