title: Orthogonal control of endogenous gene expression in mammalian cells using synthetic ligands.
abstract: Gene switches have wide utility in synthetic biology, gene therapy, and developmental biology, and multiple orthogonal gene switches are needed to construct advanced circuitry or to control complex phenotypes. Endogenous vascular endothelial growth factor (VEGF-A) is crucial to angiogenesis, and it has been shown that multiple alternately spliced VEGF-A isoforms are necessary for proper blood vessel formation. Such a necessity limits the utility of direct transgene delivery, which can provide only one splice variant. To overcome this limitation, we constructed a gene switch that can regulate the (VEGF-A) locus in mammalian cells by combining an engineered estrogen receptor (ER) ligand-binding domain (LBD), a p65 activation domain, and an artificial zinc-finger DNA binding domain (DBD). Our gene switch is specifically and reversibly controlled by 4,4'-dyhydroxybenzil (DHB), a small molecule, non-steroid synthetic ligand, which acts orthogonally in a mammalian system. After optimization of the gene switch architecture, an endogenous VEGF-A induction ratio of >100-fold can be achieved in HEK293 cells at 1 µM DHB, which is the highest endogenous induction reported to date. In addition, induction has been shown to be reversible, repeatable, and sustainable. Another advantage is that the ligand response is tunable by varying the clonal composition of a stably integrated cell line. The integration of our findings with the technology to change ligand specificity and DNA binding specificity will provide the framework for generating a wide array of orthogonal gene switches that can control multiple genes with multiple orthogonal ligands.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Liang
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McLachlan
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D005786:Gene Expression Regulation; D005812:Genes, Switch; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D009363:Neoplasm Proteins / Q000235:genetics; D010658:Phenylglyoxal / Q000031:analogs & derivatives / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: C545672:4,4'-dihydroxybenzil; D008024:Ligands; D009363:Neoplasm Proteins; D011960:Receptors, Estrogen; D042461:Vascular Endothelial Growth Factor A; D010658:Phenylglyoxal