Date of Award
Newman, Robert H.
Cell-based assays are central to life science research. In many cases, these assays require transient, or stable, transfection of an exogenous DNA of the cells under study. Previous studies examining transcriptional regulation mediated by the tumor suppressor, p53, identified several putative p53-regulated promoters involved in diepoxybutene (DEB)-induced cancer progression. However, due to potential differences in transfection efficiencies among different promoter constructs, a direct comparison of these promoters was difficult to assess. Therefore, we sought to develop a generalizable normalization method that would enable direct comparison of promoter activation regardless of transfection efficiency. Specifically, we aimed to use GFP co-transfection as a means of normalizing the signal from transient transfection of a luciferase-based reporter system in TK6 B cells. We focused primarily on cationic lipid-based transfection reagents because 1) they have been widely used to transfect a variety of cell types, 2) they cause less stress on the cells than electroporation, 3) they are safer and faster than viral transduction, and 4) they are more cost effective than specialized transfection methods, such as nucleofection. In this study, we conducted a comparative analysis of the liposome-based transfection reagents, Metafectene Pro, Lipofectamine 2000, Turbofect, and PEI, as well as nucleofection, which is an alternative transfection method based on electroporation. Co-transfection with plasmids encoding luciferase and GFP was achieved using the optimized conditions. These studies demonstrate that GFP co-transfection is an effective means of normalizing for differences in transfection efficiency using luciferase-based reporters.
Parson, II Carl, "A Generalizable Method To Normalize Luciferase Activity In Transiently Transfected B Cells" (2015). Theses. 269.