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Versatile Vectors for Ponasterone A- Inducible Control of Gene Expression in,,,Mammalian Cells

growth factors, and steroid hormones or employ synthetic promoters and inducible activators that often contain cis and trans elements derived from bacteria or yeasts.2 However, the majority of these systems have drawbacks: pleiotropic effects caused by the inducer or the transcriptional activator, prohibitively high background expression in the absence of inducer, or poor penetrance and/or clearance of the inducer in some tissues.

Stratagene has recently introduced a better system for controlled mammalian expression based on the finding that the insect molting hormone, ecdysone, stimulates transcriptional activation in mammalian cells harboring the ecdysone receptor protein from the fruit fly Drosophila melanogaster.3 The ecdysone analogs ponA and muristerone A (murA) efficiently penetrate all tissues, including the brain, due to their lipophilic nature and short in vivo half-life. This results in rapid and potent inductions and rapid clearance. Ecdysteroids are not known to affect mammalian physiology in any measurable way.

System Description

The ecdysone receptor (EcR) is a member of the RXR heterodimer family of nuclear receptors. In mammalian cells, the EcR heterodimerizes with the retinoid- X-receptor (RXR), the mammalian homologue of USP. The EcR-RXR heterodimer is capable of binding to and activating reporters that contain multiple copies of the ecdysone-responsive element (EcRE). However, because EcRE-containing reporters can be specifically trans-activated by some other lipophilic steroids, the EcR protein and EcRE recognition sequence were modified to create both a synthetic ecdysone-inducible receptor that would not bind to and trans-activate any endogenous host genes, as well as a synthetic recognition site that would not be recognized by host transcription factors.

Three amino acids in the EcR DNA-binding domain (DBD) were mutated to
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