Detection of promoter activity by flow cytometric analysis of GFP reporter expression.
Low efficiency of transfection is often the limiting factor for acquiring conclusive data in reporter assays. It is especially difficult to efficiently transfect and characterize promoters in primary human cells. To overcome this problem we have developed a system in which reporter gene expression is quantified by flow cytometry. In this system, green fluorescent protein (GFP) reporter constructs are co-transfected with a reference plasmid that codes for the mouse cell surface antigen Thy-1.1 and serves to determine transfection efficiency. Comparison of mean GFP expression of the total transfected cell population with the activity of an analogous luciferase reporter showed that the sensitivity of the two reporter systems is similar. However, because GFP expression can be analyzed at the single-cell level and in the same cells the expression of the reference plasmid can be monitored by two-color fluorescence, the GFP reporter system is in fact more sensitive, particularly in cells which can only be transfected with a low efficiency.
Ducrest
Anne-Lyse
AL
Swiss Institute for Experimental Cancer Research (ISREC), Ch. Des Boveresses 155, CH-1066 Epalinges, Switzerland.
Amacker
Mario
M
Lingner
Joachim
J
Nabholz
Markus
M
D000818:Animals; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics; D018800:Thy-1 Antigens / Q000235:genetics; D014162:Transfection / Q000379:methods
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D018800:Thy-1 Antigens; D049452:Green Fluorescent Proteins; D008156:Luciferases