A competitive RT-PCR-based approach to quantifying promoter activity of stress gene  

作　　者：MO Zhicheng, LI Xiaoyan, LI Hongfan, CHENG Xiaokuan, WU Ninghua and SHEN Yufei(National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China) 

出　　处：《Progress in Natural Science:Materials International》2002年第10期742-746,共5页自然科学进展·国际材料（英文版）

基　　金：Supported by the National Natural Science Foundation of China (Grant No. 39930050)

摘　　要：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 electrophoresis. The ratio of intensity of the two PCR bands in the same lane is taken as 'relative units of promoter efficiency' of a gene. The transcripts instead of CAT activity thus detected, may not only provide a closer look at the promoter activity of the stress gene but also normalize the efficiency of transfection simultaneously. With experimental conditions optimized, visible and reliable results can be obtained without using any sophisticated instruments. Since the results obtained with the RT-PCR approach are reproducible, much less troublesome and time-consuming, and comparable to our previous data, we thus take it as a conventional procedure for promoter activity assay of human hsp gene upon heat shock. Additionally, results from the assay have recently provided the first evidence that Rac, MEKK and JNK signals participate in the heat shock-induced expression of hsp90β gene.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 hsp90beta 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 get electrophoresis. The ratio of intensity of the two PCR bands in the same lane is taken as 'relative units of promoter efficiency' of a gene. The transcripts instead of CAT activity thus detected, may not only provide a closer look at the promoter activity of the stress gene but also normalize the efficiency of transfection simultaneously. With experimental conditions optimized, visible and reliable results can be obtained without using any sophisticated instruments. Since the results obtained with the RT-PCR approach are reproducible, much less troublesome and time-consuming, and comparable to our previous data, we thus take it as a conventional procedure for promoter activity assay of human hsp gene upon heat shock. Additionally, results from the assay have recently provided the first evidence that Rac, MEKK and JNK signals participate in the heat shock-induced expression of hsp90beta gene.

关 键 词：CHLORAMPHENICOL acetyltransferase  mRNA  PROMOTER activity  COMPETITIVE RT-PCR. 

分 类 号：R346[医药卫生—基础医学]