Optimization of a cAMP response element signal pathway reporter system.
A sensitive cAMP response element (CRE) reporter system is essential for studying the cAMP/protein kinase A/cAMP response element binding protein signal pathway. Here we have tested a few CRE promoters and found one with high sensitivity to external stimuli. Using this optimal CRE promoter and the enhanced green fluorescent protein as the reporter, we have established a CRE reporter cell line. This cell line can be used to study the signal pathway by fluorescent microscope, fluorescence-activated cell analysis and luciferase assay. This cell line's sensitivity to forskolin, using the technique of fluorescence-activated cell sorting, was increased to approximately seven times that of its parental HEK 293 cell line, which is currently the most commonly used cell line in the field for the signal pathway study. Therefore, this newly created cell line is potentially useful for studying the signal pathway's modulators, which generally have weaker effect than its mediators. Our research has also established a general procedure for optimizing transcription-based reporter cell lines, which might be useful in performing the same task when studying many other transcription-based signal pathways.
Shan
Qiang
Q
Department of Pharmacology, University of Washington, Seattle, WA 98195, USA. q.shan@uq.edu.au
Storm
Daniel R
DR
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002461:Cell Line, Transformed; D000242:Cyclic AMP / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D019426:Integrases / Q000235:genetics*; D051379:Mice; D041681:NIH 3T3 Cells; D020218:Response Elements / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; C045073:Cre recombinase; D019426:Integrases