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Nucleic Acids Research, 2002, Vol. 30, No. 14 e65
© 2002 Oxford University Press

Detection of promoter activity by flow cytometric analysis of GFP reporter expression

Anne-Lyse Ducrest, Mario Amacker, Joachim Lingner and Markus Nabholz*

Swiss Institute for Experimental Cancer Research (ISREC), Ch. Des Boveresses 155, CH-1066 Epalinges, Switzerland

*To whom correspondence should be addressed. Tel: +41 021 692 58 34; Fax: +41 021 652 69 33; Email: markus.nabholz{at}isrec.unil.ch
Present address: Mario Amacker, Gnothis SA, PSE-B EPFL, CH-1015 Lausanne, Switzerland

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.


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