Characterization of a molecular switch system that regulates gene expression in mammalian cells through a small molecule.
BACKGROUND
Molecular 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 x receptor to be responsive to the synthetic compound, LG335, but not responsive to its natural ligand, 9-cis-retinoic acid.


RESULTS
This 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 x 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.


CONCLUSIONS
We 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.
Taylor
Jennifer L
JL
School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, GA 30332, USA.
Rohatgi
Priyanka
P
Spencer
H Trent
HT
Doyle
Donald F
DF
Azizi
Bahareh
B
D000077556:Alitretinoin; D000818:Animals; D005786:Gene Expression Regulation; D006801:Humans; D008024:Ligands; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids; D011401:Promoter Regions, Genetic; D047488:Retinoid X Receptors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D019076:Transgenes; D014212:Tretinoin / Q000378:metabolism
D008024:Ligands; D047488:Retinoid X Receptors; D000077556:Alitretinoin; D014212:Tretinoin