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. 2009 Jun 12;5(1):331–338.

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This is an open access article, published under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/). This license permits non-commercial use, distribution and reproduction of the article, provided the original work is appropriately acknowledged with correct citation details.

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Figure 1. — A. Utilization of the lac-operon to generate a positive-readout reporter model for the detection of siRNA. a) In the uninduced state lac repressor (lacI) is transcribed and translated into protein (LacI), which binds lac operators (lacO) located within the promoter and an SV40 intron upstream of the luciferase gene, suppressing reporter gene expression. b) IPTG alters the conformation of LacI preventing it from binding to lacO, allowing RSV driven luciferase expression. c) siRNA specifically targeting lacI mRNA causes repressor protein levels to fall permitting RSV driven luciferase expression. B. Plasmid vectors employed in the inducible luciferase expression system. pOPRSVILuc was created by excision of the luciferase reporter gene from pGL-3 Basic by XbaI and XhoI restriction digestion and ligated into the multiple cloning site of pOPRSVI/MCS. pOPRSVI-Luc contains two lac operator binding sites (lacO₁ and lacO₂) within the RSV promoter and the SV40 intron. pCMV-LacI (a component of the LacSwitch Inducible System) expresses lac repressor from a CMV promoter; pEF1αLacI was created by replacing the CMV promoter with the human elongation factor 1 alpha (EF1α) promoter excised from pEF1αLux.G by AflII and BalII digestion.