Real-time detection of gene promoter activity: quantitation of toxin gene transcription.
We have developed a new method for quantification of promoter activity in cell lines transfected with recombinant plasmids containing the reporter gene encoding chloramphenicol acetyl transferase (CAT) by real-time PCR. As the efficiency of transfection has a direct influence on the total mRNA produced, we have used the neomycin-resistance gene present within the same vector DNA to normalize the measurement of mRNA levels. Three promoters from genes encoding toxins (pre-synaptic neurotoxin phospholipase A(2), post-synaptic alpha neurotoxin and cardiotoxin), believed to have evolved from the same ancestor but exhibiting different promoter activities, have been employed in this study to demonstrate the feasibility and accuracy of the method in CAT gene reporter analysis.
Jeyaseelan
K
K
Department of Biochemistry, Faculty of Medicine, National University of Singapore, 10 Medical Drive, 119260, Singapore. bchjeya@nus.edu.sg
Ma
D
D
Armugam
A
A
D000818:Animals; D016466:CHO Cells; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004179:Cobra Cardiotoxin Proteins / Q000235:genetics; D006224:Cricetinae; D004351:Drug Resistance / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000235:genetics; D010741:Phospholipases A / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D013997:Time Factors; D014118:Toxins, Biological / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection
D004179:Cobra Cardiotoxin Proteins; D008565:Membrane Proteins; D009498:Neurotoxins; D012333:RNA, Messenger; D014118:Toxins, Biological; D015500:Chloramphenicol O-Acetyltransferase; D010741:Phospholipases A