TAILIEUCHUNG - Báo cáo khoa học: Improved ecdysone receptor-based inducible gene regulation system

To develop an ecdysone receptor (EcR)-based inducible gene regulation system, several constructs were prepared by fusing DEF domains of Choristoneura fumiferanaEcR (CfEcR),C. fumiferanaultraspiracle (CfUSP),Mus muscu-lus retinoid X receptor (MmRXR) to either GAL4 DNA binding domain (DBD) or VP16 activation domain. These constructs were tested in mammalian cells to evaluate their ability to transactivate luciferase gene placed under the control of GAL4 response elements and synthetic TATAA promoter. . | Eur. J. Biochem. 270 1308-1315 2003 FEBS 2003 doi Improved ecdysone receptor-based inducible gene regulation system Subba R. Palli1 Mariana Z. Kapitskaya2 Mohan B. Kumar2 and Dean E. Cress2 1 Department of Entomology College of Agriculture University of Kentucky KY USA 2RHeoGene LLC Spring House PA USA To develop an ecdysone receptor EcR -based inducible gene regulation system several constructs were prepared by fusing DEF domains of Choristoneura fumiferana EcR CfEcR C. fumiferana ultraspiracle CfUSP Mus muscu-lus retinoid X receptor MmRXR to either GAL4 DNA binding domain DBD or VP16 activation domain. These constructs were tested in mammalian cells to evaluate their ability to transactivate luciferase gene placed under the control of GAL4 response elements and synthetic TATAA promoter. A two-hybrid format switch where GAL4 DBD was fused to CfEcR DEF and VP16 AD was fused to MmRXR EF was found to be the best combination. It had the lowest background levels of reporter gene activity in the absence of a ligand and the highest level of reporter gene activity in the presence of a ligand. Both induction and turnoff responses were fast. A 16-fold induction was observed within 3 h of ligand addition and increased to 8942-fold by 48 h after the addition of ligand. Withdrawal of the ligand resulted in 50 and 80 reduction in reporter gene activity by 12 h and 24 h respectively. Keywords gene switch ponasterone A receptors EcR RXR. Twenty hydroxyecdysone 20E is a steroid hormone that regulates molting metamorphosis reproduction and various other developmental processes in insects. Ecdysone functions through a heterodimeric receptor complex comprised of ecdysone receptor EcR and ultraspiracle USP . Both EcR and USP cDNAs have been cloned from Drosophila melanogaster and several other insects 1 and were shown to be members of the steroid hormone receptor superfamily. Members of this superfamily are characterized by the presence of five modular

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