A3 超音波で活性化するプロモーターの構築

抄録

Promoters responsive to sonication could be useful for efficient gene therapy since, using such a promoter, expression of a therapeutic gene could be controlled by sonication. Synthesized DNA oligomers containing binding motifs of transcription factors, AP-1, NF-kB, NF-Y and CBF-A, which are activated by oxidative stress, were randomly ligated them and linked to a TATA box sequence to construct artificial promoters. These promoters were introduced upstream of the luciferase gene to control its expression. A plasmid with such a gene cassette was transfected into HeLa cells and sonicated with 1MHz ultrasound at 1W/cm^2 and 10% duty factor for 60 sec. Transfected cells with two out of six plasmids significantly increased luciferase activities compared with each of unsonicated controls 6 h after sonication. Enhancement with clone #11 showing the highest increase turned out to be dependent on intensity and duration of sonication. Nucleotide sequence analysis indicated the promoter was 214 bp long, composed of 16 copies of transcription factor binding motifs. To improve reactivity of clone #11 to sonication, random mutations were introduced by error prone PCR. Two series of PCR reactions on clone #11 resulted in a new promoter over 4 fold more reactive to sonication.