title: Regulation of endogenous gene expression with a small-molecule dimerizer.
abstract: Artificial transcription factors containing designer zinc-finger DNA-binding domains (DBDs) have been used to activate or repress expression of a growing number of endogenous genes. We have combined targeted zinc-finger DBD technology with a dimerizer-regulated gene expression system to permit the small-molecule control of endogenous gene transcription. We constructed a dimerizer-responsive transcription factor that incorporates an artificial zinc-finger DBD targeted to the promoter of the human VEGF gene. Introduction of this activator into human cells allowed expression of the chromosomal VEGF gene to be induced by a small-molecule dimerizer compound consisting of a nonimmunosuppressive rapamycin analog. We found that by directly regulating zinc-finger protein (ZFP) activity, we could circumvent difficulties encountered in the generation of cell lines stably expressing conventional unregulated activators. Dimerizer-dependent VEGF induction was rapid, tight, and dose dependent, and resulted in VEGF protein expression levels several-fold greater than those produced by the natural hypoxic response.
authors.affiliation: ARIAD Gene Therapeutics, Inc., 26 Landsdowne Street, Cambridge, MA 02139, USA. roy.pollock@ariad.com
authors.forename: Roy
authors.initials: R
authors.lastname: Pollock
authors.forename: Maryann
authors.initials: M
authors.lastname: Giel
authors.forename: Katja
authors.initials: K
authors.lastname: Linher
authors.forename: Tim
authors.initials: T
authors.lastname: Clackson
mesh_terms: D002460:Cell Line; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D016228:Endothelial Growth Factors / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics*; D007668:Kidney / Q000196:embryology; D008222:Lymphokines / Q000096:biosynthesis / Q000235:genetics*; D008957:Models, Genetic; D010957:Plasmids; D020123:Sirolimus / Q000008:administration & dosage*; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D020123:Sirolimus