title: Positive feedback system provides efficient and persistent transgene expression.
abstract: The two-step transcriptional amplification (TSTA) system, using artificial transcription factors, effectively enhances transgene expression. In this study, a TSTA system-based positive feedback system was developed to achieve efficient and persistent transgene expression. A fusion protein of the sequence-specific DNA binding domain of yeast GAL4 and the transcriptional activation domain of herpes simplex virus VP16 (GAL4-VP16) was used as an "activator" to amplify the expression of the luciferase "reporter" gene. It was found that the introduction of five tandem copies of the GAL4 recognition sequence (G5) into both the upstream and downstream regions of the expression cassette synergistically enhanced the transgene expression. The upstream and downstream G5 sequences were introduced into the expression cassette of the activator itself, and into that of the reporter, to form the positive feedback loop that enabled continuous activator expression. This positive feedback system maintained the expression levels of the reporter for 4 days in HeLa cells and for a week in mouse liver, while those from the usual plasmids decreased by 30- and 50-fold, respectively. These results constitute the first evidence that the positive feedback system is a useful method for long-term transgene expression in cultured cells and in vivo. This system would be applicable to gene therapy, in vivo imaging, and biotechnology.
authors.affiliation: Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ochiai
authors.forename: Hideyoshi
authors.initials: H
authors.lastname: Harashima
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kamiya
mesh_terms: D000818:Animals; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics; D005260:Female; D017930:Genes, Reporter / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D019076:Transgenes / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; C070633:Gal-VP16; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D008156:Luciferases