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Updated: Jul 24, 2026

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides
Published on: November 29, 2016
A single point mutation in ecdysone receptor leads to increased ligand specificity: implications for gene switch
M B Kumar1, T Fujimoto, D W Potter
1RheoGene LLC, P.O. Box 949, 727 Norristown Road, Spring House, PA 19477, USA.
Abstract:
The ecdysone receptor (EcR), a member of the nuclear receptor superfamily, plays an important role in regulating development and reproduction in insects. The EcR binds to ecdysteroids and regulates transcription of genes that contain ecdysone response elements. The EcR has been used to develop inducible gene switches for efficient regulation of foreign genes in applications such as gene therapy, protein production, and functional genomics. An EcR [Choristoneura fumiferana EcR (CfEcR)] homology model was constructed, and 17 amino acid residues were identified as critical for 20-hydroxyecdysone binding. Mutation of these amino acids followed by analysis of these mutants in transactivation (in insect and mammalian cells and in vivo in mice) and ligand-binding assays identified one particular mutant (A110P) that failed to respond to steroids, but its response to the diacylhydrazine nonsteroidal ligands RG-102240 (GS(TM)E) and RG-102317 was unaffected. This steroid-insensitive EcR mutant has potential gene switch applications in insects and plants that have endogenous ecdysteroids. In addition, this mutant would be also useful for developing orthogonal EcR-ligand pairs for simultaneous regulation of multiple genes in the same cell.
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