title: Effects of endogenous proteins and microRNA target sequence in a positive feedback system.
abstract: A positive feedback system, using GAL4-vp16 (a fusion protein of yeast GAL4 and herpes simplex virus vp16) as an activator and firefly luciferase as a reporter, maintained luciferase expression for 7 d in mice. However, the luciferase expression decreased after 7 d, and this phenomenon could be caused by immunoreactions against these exogenous proteins. This hypothesis was examined by the following three strategies, designed to avoid the putative immunoreactions: (i) use of the endogenous secreted alkaline phosphatase (SEAP) protein as a reporter, (ii) replacement of vp16 with endogenous transcription factors, and (iii) insertion of the target sequence of microRNA expressed in cells of hematopoietic origin, to suppress GAL4-vp16 expression in antigen-presenting cells. The results obtained in this study suggested that silencing would be induced by mechanism(s) besides immunoreactions against reporter and activator proteins.
authors.affiliation: Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
authors.forename: Genki N
authors.initials: GN
authors.lastname: Kanda
authors.forename: Ryohei
authors.initials: R
authors.lastname: Togashi
authors.forename: Hideyoshi
authors.initials: H
authors.lastname: Harashima
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kamiya
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000938:Antigen-Presenting Cells; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D025461:Feedback, Physiological; D005260:Female; D048888:Fireflies; D015870:Gene Expression / Q000276:immunology; D017930:Genes, Reporter; D006412:Hematopoietic Stem Cells; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D035683:MicroRNAs; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015533:Transcriptional Activation / Q000276:immunology; D019076:Transgenes / Q000276:immunology
chemical_list: D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; C070633:Gal-VP16; D017405:Herpes Simplex Virus Protein Vmw65; D035683:MicroRNAs; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D008156:Luciferases; D000469:Alkaline Phosphatase