(405e) Using An Orthogonal Ligand-Receptor Pair to Control Endogenous Gene Expression In Mammalian Cell

Small molecule regulated gene expression systems (gene switches) are powerful tools for gene therapy, functional genomics, metabolic engineering, and tissue engineering. By combining an engineered estrogen receptor ligand-binding domain (LBD), an activation domain, and an artificial zinc-finger DNA-binding domain (DBD), we have constructed a gene switch that is able to regulate the endogenous VEGF-A (vascular endothelial growth factor A) expression in mammalian cell. The 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, a VEGF-A induction ratio of ~200X can be achieved in HEK293 cells at micromolar concentration of DHB. This gene switch can potentially be combined with other artificial DBDs to control other endogenous genes. Furthermore, we can also substitute the LBD with one from another orthogonal ligand-receptor pair to create gene switches that can simultaneously regulate multiple genes in a mammalian system.