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Chloramphenicol acetyltransferase (CAT) as a stable reporter has been broadly adopted to detect promoter activity of many stress genes in eukaryotic cells. A simplified, convenient and relatively accurate assay to reveal CAT activities driven by heat shock gene promoter upon cell stress is reported here. A CAT mutant control plasmid pMCAT is constructed out of pBLCAT3, with a DNA segment within the CAT gene deleted. The control plasmid pMCAT along with the wild type CAT reporter gene driven by the regulatory sequence of hsp90β gene are co-transfected into Jurkat cells. Following total cellular RNA extracted and reverse transcribed, PCR is carried out to amplify two individual fragments with the same pair of synthesized primers, which bind to the identical sequences in both pMCAT and the CAT reporter plasmids. The two PCR fragments amplified from the reporter and control RNAs are identified by their size differences in agarose gel electropboresis. The ratio of intensity of the two PCR bands in the same
Chloramphenicol acetyltransferase (CAT) as a stable reporter has been broadly adopted to detect promoter activity of many stress genes in eukaryotic cells. A simplified, convenient and relatively accurate assay to reveal CAT activities driven by heat shock gene promoter upon cell stress is reported here. A CAT mutant control plasmid pMCAT is constructed out of pBLCAT3, with a DNA segment within the CAT gene deleted. The control plasmid pMCAT along with the wild type CAT reporter gene driven by the regulatory sequence of hsp90β gene are co-transfected into Jurkat cells. PCR was carried out to amplify two individual fragments with the same pair of synthesized primers, which bind to the identical sequences in both pMCAT and the CAT reporter plasmids. The two PCR fragments amplified from the reporter and control RNAs are identified by their size differences in agarose gel electropboresis. The ratio of intensity of the two PCR bands in the same