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ARS Home » Midwest Area » Lexington, Kentucky » Forage-animal Production Research » Research » Publications at this Location » Publication #193224
Title: Development of a tightly regulated and highly inducible ecdysone receptor gene switch for plants through the use of retinoid X receptor chimeras

Author
item TAVVA, VENKATA - UNIVERSITY OF KENTUCKY
item Dinkins, Randy
item PALLI, S - UNIVERSITY OF KENTUCKY
item COLLINS, GLENN - UNIVERSITY OF KENTUCKY

Submitted to: Transgenic Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/10/2006
Publication Date: 9/4/2007
Citation: Tavva, V.S., Dinkins, R.D., Palli, S.R., Collins, G.B. 2007. Development of a tightly regulated and highly inducible ecdysone receptor gene switch for plants through the use of retinoid X receptor chimeras. Transgenic Research. 16:559-612.

Interpretive Summary: Chemical inducible gene regulation systems provide essential tools for the precise regulation of transgene expression in plants and animals. We have recent developed a two-hybrid ecdysone receptor (EcR) gene regulation system that works in conjunction with the retinoid X receptor of Homo sapiens (HsRXR) and insect, Locusta migratoria RXR (LmRXR). To further improve some of the observed drawbacks in the original two-hybrid interactions , chimeras between Homo sapiens retinoid X receptor (HsRXR) and insect, Locusta migratoria RXR (LmRXR) were tested in tobacco protoplasts as partners with Choristoneura fumiferana EcR (CfEcR) in inducing expression of the luciferase reporter gene. The RXR chimera 9 (CH9) along with CfEcR, in a two-hybrid format gave the best results in terms of low background expression levels in the absence of ligand and high induced expression levels of the reporter gene in the presence of low concentrations of methoxyfenozide as an inducer. The performance of CH9 was further tested in corn and soybean protoplasts and the data obtained was compared with the other EcR switches that contained the wild-type LmRXR or HsRXR as EcR partners. In both transient expression studies and stable transformation experiments, the fold induction values obtained with the CH9 switch were several times higher than the values obtained with the other EcR switches containing LmRXR or HsRXR. The new CfEcR two-hybrid gene switch that uses the RXR chimera 9 as a partner in inducing reporter gene expression provides an efficient, ligand-sensitive and tightly regulated gene switch for plants.

Technical Abstract: Chemical inducible gene regulation systems provide essential tools for the precise regulation of transgene expression in plants and animals. Recent development of a two-hybrid ecdysone receptor (EcR) gene regulation system has solved some of the drawbacks that were associated with the monopartate gene switch. To further improve the versatility of the two-hybrid EcR gene switch for its wide spread use in plants, chimeras between Homo sapiens retinoid X receptor (HsRXR) and insect, Locusta migratoria RXR (LmRXR) were tested in tobacco protoplasts as partners with Choristoneura fumiferana EcR (CfEcR) in inducing expression of the luciferase reporter gene. The RXR chimera 9 (CH9) along with CfEcR, in a two-hybrid format gave the best results in terms of low background expression levels in the absence of ligand and high induced expression levels of the reporter gene in the presence of nanomolar concentrations of methoxyfenozide ligand. The performance of CH9 was further tested in corn and soybean protoplasts and the data obtained was compared with the other EcR switches that contained the wild-type LmRXR or HsRXR as EcR partners. In both transient expression studies and stable transformation experiments, the fold induction values obtained with the CH9 switch were several times higher than the values obtained with the other EcR switches containing LmRXR or HsRXR. The new CfEcR two-hybrid gene switch that uses the RXR chimera 9 as a partner in inducing reporter gene expression provides an efficient, ligand-sensitive and tightly regulated gene switch for plants.