TAILIEUCHUNG - Báo cáo khoa học: Functional characterization of ecdysone receptor gene switches in mammalian cells

Regulated expression of transgene is essential in basic research as well as for many therapeutic applications. The main purpose of the present study is to understand the functioning of the ecdysone receptor (EcR)-based gene switch in mammalian cells and to develop improved versions of EcR gene switches. | ễFEBS Journal Functional characterization of ecdysone receptor gene switches in mammalian cells Siva K. Panguluri1 Prasanna Kumar2 and Subba R. Palli1 1 Department of Entomology College of Agriculture University of Kentucky Lexington KY USA 2 RheoGene Inc. Norristown PA USA Keywords ChIP assay EcR gene switch RXR RNAi Correspondence S. R. Palli Department of Entomology College of Agriculture University of Kentucky Lexington KY 40546 USA Fax 1 859 323 1120 Tel 1 859 257 4962 E-mail RPALLI@ Received 15 August 2006 revised 5 October 2006 accepted 18 October 2006 doi Regulated expression of transgene is essential in basic research as well as for many therapeutic applications. The main purpose of the present study is to understand the functioning of the ecdysone receptor EcR -based gene switch in mammalian cells and to develop improved versions of EcR gene switches. We utilized EcR mutants to develop new EcR gene switches that showed higher ligand sensitivity and higher magnitude of induction of reporter gene expression in the presence of ligand. We also developed monopartite versions of EcR gene switches with reduced size of the components that are accommodated into viral vectors. Ligand binding assays revealed that EcR alone could not bind to the nonsteroidal ligand RH-2485. The EcR s heterodimeric partner ultraspiracle is required for efficient binding of EcR to the ligand. The essential role of retinoid X receptor RXR or its insect homolog ultraspiracle in EcR function is shown by RXR knockdown experiments using RNAi. Chromatin immunoprecipitation assays demonstrated that VP16 activation domain AD GAL4 DNA binding domain DBD EcR ligand binding domain LBD or GAL4 DBD EcR LBD fusion proteins can bind to GAL4 response elements in the absence of ligand. The VP16 AD fusion protein of a chimera between human and locust RXR could heterodimerize with GAL4 DBD EcR LBD in the absence of ligand but the VP16 AD fusion protein of Homo .

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