Abstract
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.
Highlights
Molecular switch systems that activate gene expression by a small molecule are effective technologies that are widely used in applied biological research
Doyle et al showed that a retinoid × receptor (RXR) variant consisting of three mutations in the ligand binding domain (LBD), Q275C, I310M, F313I (QCIMFI), and a unchanged DNA binding domain (DBD) activated transcription in response to LG335 but not by the RXR natural ligand, 9-cis-retinoic acid (9cRA) [29]
QCIMFI is activated in response to LG335 at concentrations as low as 100 nM (EC50 value is 38 nM) with a 14.5 ± 1.6-fold induction and no activation with 9cRA (Figure 1A)
Summary
Molecular switch systems that activate gene expression by a small molecule are effective technologies that are widely used in applied biological research. Upon the addition of RU486, the regulator binds a DNA sequence composed of six Gal response elements (RE) and activates gene expression. One disadvantage of this system is that the ligand, RU486, has been shown to interfere with other biological pathways (at a much higher concentration of ligand), so long term usage could have extensive side effects [9]. In the tetracycline (Tet) inducible system, the prokaryotic protein, Tet, binds to a specific DNA sequence called tetO in response to tetracycline or doxycycline (dox) [4] This system can function as both an ON-switch as well as an OFF-switch [10]. The concerns posed by these systems permit the development of new or improved molecular switch systems
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