The luciferase assay is a very sensitive and convenient way to examine the transcriptional activity of a gene. Typically, a promoter region containing 2 to 3 kb of a 50 flanking sequence and part of the first exon is cloned in a commercial vector PGL3-basic vector (Promega) to obtain a photinus lucif-erase gene driven by the promoter of the target gene. PCR amplification of the genomic DNA is an easy way to obtain the promoter fragment. The promoter can be analyzed as described earlier for the TonEBP-driven luciferase reporter gene. A clear response to hypertonicity suggests the involvement ofTonEBP. The next step is to make mutant versions of the promoter-reporter with inactive TonE. This is best achieved by mutating GG to AA in candidate TonEBP sites: TGGAAAxxYnY (Y is C or T). Loss of the response to
Was this article helpful?