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| Construction and Verification of an Inducible EMT Model in Mouse Melanoma Stably Overexpressing Snail |
| FANG Rui, GUO Qiang, DU Jun |
| School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China |
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Abstract Objective: To establish an inducible EMT cell model which stably overexpressing recombinant mouse Snail and evaluate its application in fluorescence imaging in vitro and in vivo. Methods: The mouse Snail gene was amplified from pCMV6-mSnail by PCR and cloned into eukaryotic expression plasmid pL-tdTomato-Neo. The recombinant colonies were identified by double enzyme digestion and DNA sequencing. The mouse melanoma B16 cells were transfected with recombinant plasmid pL-tdTomato-mSnail and the stably transfected cells B16/Td-mSN were selected with culture media supplemented with G418. The mRNA and protein levels of Snail as well as EMT markers E-cadherin and Vimentin were examined by qPCR and Western blot. Also, the B16 and B16/Td-mSN cells were inoculated subcutaneously into the nude mice respectively to prepare allografted tumor model and examined by fluorescence imaging system. Results: The recombinant expression plasmid pL-tdTomato-mSnail was constructed correctly, in which the red fluorescence protein tdTomato, mouse Snail and resistance gene were seriesly connected by two 2A peptides. The selected B16/dT-mSN cells express high levels of Snail and red fluorescence protein. Also, the cells display elongated and spindle-like morphology as well as down-regulation of E-cadherin and up-regulation of Vimentin. The allograft mouse model for in vivo imaging was successfully established by use of this stable cell line. And the fluorescence signal was stable and strong. Conclusion: An EMT cell model stably expressing recombinant mouse Snail and red fluorescence protein was successfully established. The cell model provides a convenient, intuitive and stable tool for imaging studies of Snail biological function during tumor EMT progress.
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Received: 25 March 2013
Published: 25 July 2013
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