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In vitro promoter strength analysis by expressional studies of transgene driven by both EGP2 and CMV promoter in cancerous and normal cell lines.

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posted on 31.12.2013 by Ravikanth Danda, Gopinath Krishnan, Kalaivani Ganapathy, Uma Maheswari Krishnan, Khetan Vikas, Sailaja Elchuri, Nivedita Chatterjee, Subramanian Krishnakumar

EpCAM expression was evaluated by western blot analysis and β-actin was used as a loading control (A). The transgene expression driven by EGP2 and CMV promoter was evaluated by transient transfection of CMV-TK, EGP2-TK, CMV-luc, and EGP2-luc. After 2 days of transfection TK protein expression driven by CMV promoter was evaluated by western blot analysis and the results were normalized against the β-actin (B). TK protein expression driven by EGP2 promoter was evaluated by western blot analysis and results were normalized against the β-actin (C). Protein quantification was quantified by using ImageJ software and protein expression was expressed in relative signal intensity values. TK mRNA levels were quantified by Real-time PCR, the mRNA levels were normalized against the GAPDH and were expressed in relative fold change units (D). Luciferase expression analysis was performed by quantifying the secreted Gaussia luciferase. The results were normalized against un-transfected cells and were expressed in relative light units (E). Normalized levels of luciferase expression as percentage shown as mean ± S.D (n = 3). *p<0.05, **p<0.01, ***p<0.001 vs. Control, +p<0.05, ++p<0.01, +++p<0.001 vs. CMV-luc.

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