| Popis: |
The ecdysone receptor (EcR)-based gene switch is used to regulate gene expression in a ligand-dependent manner. Gene switches may have utility in human gene therapies that require a specifically regulated level of gene expression, where it is desirable to terminate the therapy when it is no longer needed, or where continuation of the therapy would be unsafe. In addition, multiplexed gene switches allow for the differential regulation of several genes simultaneously. These EcR-based switches are comprised of EcR fused to a GAL4 DNA binding domain, and a chimera of human and insect retinoid X receptor fused to a VP16 activation domain (pVP16-Hs-LmRXR), which regulates a reporter gene that is placed under the control of a 6GAL4 response element and a transthyretin minimal promoter (p6xGAL4RE-TTR-SEAP). Three multiplex gene switches have been developed by mutating the EcR ligand-binding domain in a site-specific manner. Each switch shows specificity for either diacylhydrazines (DAH), tetrahydroquinolines (THQ) or ecdysteroid (EcD) ligands, and are not cross-activated by the other chemotypes. In mouse electroporation studies, a switch based on a V96T/N119F double mutant of EcR showed high specificity for EcD (Ponasterone A, PonA) but was completely refractory toward both DAH (GSTM-E) and THQ (RG-120499) ligands. Likewise, another double mutant of EcR, V128F/A110P, showed specificity toward THQ, but was refractory toward DAH (GSTM-E) and EcD (PonA) ligands. A third gene switch that uses A110P mutant of EcR was sensitive to GSTM-E ligand but not to either EcD (PonA) or THQ (RG-120499) ligands. A dual switch containing V96T/N119F and V128F/A110P EcR double mutants were used to demonstrate the simultaneous regulation of two genes in vivo. |
