Characterization of a molecular switch system that regulates gene expression in mammalian cells through a small molecule

BMC Biotechnology

Research Article

H Trent Spencer¹  Priyanka Rohatgi²  Jennifer L Taylor³  Bahareh Azizi³  Donald F Doyle³ 

[1] Aflac Cancer Center and Blood Disorders Services, Department of Pediatrics, Emory University School of Medicine, 30322, Atlanta, GA, USA;Pharmacokinetics and ADME group, Baxter Healthcare, One Baxter Parkway Deerfield, 60015-4625, IL, USA;School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive, 30332, Atlanta, GA, USA;

关键词: HEK293T Cell;    NIH3T3 Cell;    Internal Ribosomal Entry Site;    Molecular Switch;    Bexarotene;   

DOI  :  10.1186/1472-6750-10-15

 received in 2009-10-09, accepted in 2010-02-18,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundMolecular switch systems that activate gene expression by a small molecule are effective technologies that are widely used in applied biological research. Nuclear receptors are valuable candidates for these regulation systems due to their functional role as ligand activated transcription factors. Previously, our group engineered a variant of the retinoid × receptor to be responsive to the synthetic compound, LG335, but not responsive to its natural ligand, 9-cis-retinoic acid.ResultsThis work focuses on characterizing a molecular switch system that quantitatively controls transgene expression. This system is composed of an orthogonal ligand/nuclear receptor pair, LG335 and GRQCIMFI, along with an artificial promoter controlling expression of a target transgene. GRQCIMFI is composed of the fusion of the DNA binding domain of the yeast transcription factor, Gal4, and a retinoid × receptor variant. The variant consists of the following mutations: Q275C, I310M, and F313I in the ligand binding domain. When introduced into mammalian cell culture, the switch shows luciferase activity at concentrations as low as 100 nM of LG335 with a 6.3 ± 1.7-fold induction ratio. The developed one-component system activates transgene expression when introduced transiently or virally.ConclusionsWe have successfully shown that this system can induce tightly controlled transgene expression and can be used for transient transfections or retroviral transductions in mammalian cell culture. Further characterization is needed for gene therapy applications.

【 授权许可】

Unknown   
© Taylor et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.