Manipulation of gene expression by an ecdysone-inducible gene switch in tumor xenografts

Karns, Larry R.; Kisielewski, Anne; Gulding, Kathryn M; Theodorescu, Dan

doi:10.1186/1472-6750-1-11

Cited by 19 publications

(15 citation statements)

References 18 publications

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“…Both induction and switch‐off responses were rapid. Recently, our collaborators confirmed the performance of this switch in stable cell lines as well as in mouse tumors [35]. Bisacylhydrazine nonsteroidal ligands have undergone an extensive battery of toxicology tests for EPA registration as commercial insecticides [33].…”

Section: Discussionmentioning

confidence: 99%

Improved ecdysone receptor‐based inducible gene regulation system

Palli

Kapitskaya

Kumar

et al. 2003

European Journal of Biochemistry

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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 musculus 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.

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Section: Discussionmentioning

confidence: 99%

Improved ecdysone receptor‐based inducible gene regulation system

Palli

Kapitskaya

Kumar

et al. 2003

European Journal of Biochemistry

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“…In summary, we provide a powerful approach to stably and inducibly suppress gene expression in mammalian cells. Given the success of the ecdysone-based inducible systems in animal models (15,33) and germ-line transmission of RNAi (34), it is conceivable to generate transgenic animals inducibly expressing RNAi. A considerable improvement to the existing design would be to obtain tissue-specific regulation in vivo by either expressing the GAL4 activator under the control of a tissue-specific promoter or by using a ''caged ecdysteroid'' (35).…”

Section: Stable and Efficient Rnai-mediated Inducible Suppression Of mentioning

confidence: 99%

“…Furthermore, doxycyclin is linked to toxicity problems, a major disadvantage for studies involving both cultured cells and animals. Ecdysone is more suitable for use in vivo because it is a naturally occurring lipophilic steroid that can penetrate tissues and is quickly metabolized and cleared (33).…”

Section: Stable and Efficient Rnai-mediated Inducible Suppression Of mentioning

confidence: 99%

Inducible, reversible, and stable RNA interference in mammalian cells

Gupta

Schoer

Egan

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

201

134

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RNA interference is a powerful genetic approach for efficiently silencing target genes. The existing method of gene suppression by the constitutive expression of short hairpin RNAs (shRNAs) allows analysis of the consequences of stably silencing genes but limits the analysis of genes essential for cell survival, cell cycle regulation, and cell development. We have developed an inducible U6 promoter for synthesis of shRNAs in both human and murine cells. Cells containing stably integrated shRNA expression constructs demonstrate stringent dosage-and time-dependent kinetics of induction with undetectable background expression in the absence of the inducer ecdysone. Inducible suppression of human p53 in glioblastoma cells shows striking morphological changes and defects in cell cycle arrest caused by DNA damage, as expected. Remarkably, the inducibility is reversible after withdrawal of the inducer, as observed by reappearance of the protein and a restoration of the original cell phenotype. Inducible and reversible regulation of RNA interference has broad applications in the areas of mammalian genetics and molecular therapeutics.

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“…Over the past years, four main systems for in vivo transgene regulation have emerged from animal studies: the tetracycline 8,9 and rapamycin-inducible systems, 10 the mammalian steroid receptor (tamoxifen and mifepristone) 11,12 and the insect steroid receptor (ecdysteroid) system. 13 Of these, the tetracycline inducible system has been the most commonly studied and is at this moment considered the most potent and clinically relevant. 14 As its original development, 8 where transgene expression was repressed in the presence of the inducer drug tetracycline (so called tet-off), several major improvement in the tetracycline inducible system have readied this system for clinical translation.…”

Section: Introductionmentioning

confidence: 99%

Developing a potentially immunologically inert tetracycline-regulatable viral vector for gene therapy in the peripheral nerve

et al. 2014

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Viral vector-mediated gene transfer of neurotrophic factors is an emerging and promising strategy to promote the regeneration of injured peripheral nerves. Unfortunately, the chronic exposure to neurotrophic factors results in local trapping of regenerating axons or other unwanted side effects. Therefore, tight control of therapeutic gene expression is required. The tetracycline/doxycycline-inducible system is considered to be one of the most promising systems for regulating heterologous gene expression. However, an immune response directed against the transactivator protein rtTA hampers further translational studies. Immunogenic proteins fused with the Gly-Ala repeat of the Epstein-Barr virus Nuclear Antigen-1 protein have been shown to successfully evade the immune system. In this article, we used this strategy to demonstrate that a chimeric transactivator, created by fusing the Gly-Ala repeat with rtTA and embedded in a lentiviral vector (i) retained its transactivator function in vitro, in muscle explants, and in vivo following injection into the rat peripheral nerve, (ii) exhibited a reduced leaky expression, and (iii) had an immune-evasive advantage over rtTA as shown in a novel bioassay for human antigen presentation. The current findings are an important step toward creating a clinically applicable potentially immune-evasive tetracycline-regulatable viral vector system.

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Manipulation of gene expression by an ecdysone-inducible gene switch in tumor xenografts

Cited by 19 publications

References 18 publications

Improved ecdysone receptor‐based inducible gene regulation system

Improved ecdysone receptor‐based inducible gene regulation system

Inducible, reversible, and stable RNA interference in mammalian cells

Developing a potentially immunologically inert tetracycline-regulatable viral vector for gene therapy in the peripheral nerve

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