[4] Comparison of enhanced green fluorescent protein and its destabilized form as transcription reporters

This chapter describes the utility of green fluorescent protein (GFP) as a reporter gene in the study of gene expression. GFP emits fluorescent light on excitation, without the addition of substrate or cofactor, both in vivo and in vitro. The GFP fluorescence activity can be detected using a fluorescence microscope, fluorometer, fluorescence-activated cell sorting (FACS) machine, or imaging microplate reader. Enhanced GFP (EGFP) is a GFP mutant with brighter fluorescence that makes the detection much more sensitive. Use of GFP as a reporter gene offers a number of advantages. These include real-time analysis, minimal sample handing, the possibility of large-quantity analysis, and high sensitivity. However, GFP is a stable protein, so that it is easily accumulated when expressed in cells. The accumulation makes the inducible expression of the reporter insensitive to any change in induction and thus it would be difficult to use in kinetics studies. This chapter discusses the utility of EGFP and destabilized EGFP (dEGFP) as transcription reporters by fusing them with NF-KB-binding sequence and thymicline kinase (TK) promoter, and comparing the difference in expression between EGFP and dEGFP. It discusses that both EGFP and dEGFP can be used as reporters in transcription studies. Also, dEGFP is more sensitive in response to changes in tumor necrosis factor (TNF) treatment owing to its faster turnover rate.