Mammalian cis-reporting plasmid may alter activities due to the derivation of host Escherichia coli strains.
Methylation status of CpG dinucleotides in the promoter/regulatory region contributes to regulation of transcriptional activities of downstream genes. Nearly all plasmid vectors used in mammalian cells are generated from transformed Escherichia coli. However, these E. coli hosts may have different DNA methylation activities. For instance E. coli JM109 and DH5alpha contain Dam and Dcm methylases, which are absent in E. coli JM110 and GM2163. It has not been determined whether plasmids propagated from E. coli of different methylation activities result in altered expression in mammalian cells when transient transfection is conducted. In this report, cis-reporting plasmids were tested. When promoter/enhancer of tested plasmids contained several Dam/Dcm sites, the cis-reporting activity was 2 to 3 fold lower for those plasmids isolated from JM109 than from JM110. In contrast, the E. coli-derived methylation had little effect on transcription when the sites of methylation resided in the coding region. These findings suggest that cis-reporting plasmids used in comparative or successive experiments are required to be derived from the E. coli strain of the same methylation status. The plasmid for promoter-transcription factor studies should be Dam/Dcm negative E. coli strain.
Lai
Ying-Fei
YF
Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan 11221, ROC.
Tseng
Yu-Ju
YJ
Yang
Feng-Yuan
FY
Au
Lo-Chun
LC
D001483:Base Sequence; D002460:Cell Line; D019175:DNA Methylation; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification; D011401:Promoter Regions, Genetic / Q000235:genetics; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D008156:Luciferases; D001616:beta-Galactosidase