Transfection Considerations

Each different SEAP construct should be transfected (and subsequently assayed) in triplicate to minimize variability among treatment groups. The primary sources of such variability are differences in transfection efficiencies. When monitoring the effect of promoter and enhancer sequences on gene expression, it is critical to include an internal control that will distinguish differences in the level of transcription from variability in the efficiency of transfection. This is easily done by cotransfecting with a second plasmid that constitutively expresses an activity which can be clearly defined from SEAP. The level of the second enzymatic activity can then be used to normalize the levels of SEAP among different treatment groups. Reporter proteins frequently used for this purpose include E coli P-galactosidase, which is expressed intracellularly, and human growth hormone (hGH), which is secreted (5).

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