23 February 2012 Inducible expression of photoacoustic reporter gene tyrosinase in cells using a single plasmid
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We have previously demonstrated that tyrosinase is a reporter gene for photoacoustic imaging since tyrosinase is the rate-limiting step in the synthesis of melanin, a pigment capable of producing strong photoacoustic signals. We previously created a cell line capable of inducible tyrosinase expression (important due to toxicity of melanin) by stably transfecting tyrosinase in MCF-7 Tet-OnR cell line (Clontech) which expresses a doxycycline-controlled transactivator. Unfortunately, Clontech provides few Tet-On Advanced cell lines making it difficult to have inducible tyrosinase expression in cell lines not provided by Clontech. In order to simplify the creation of cell lines with inducible expression of tyrosinase, we created a single plasmid that encodes both the transactivator as well as tyrosinase. PCR was used to amplify both the transactivator and tyrosinase from the Tet-OnR Advanced and pTRE-Tight-TYR plasmids, respectively. Both PCR products were cloned into the pEGFP-N1 plasmid and the newly created plasmid was transfected into ZR-75-1, MCF-7, and MIA PaCa-1 cells using lipofectamine. After several days, brown melanin was only observed in cells incubated with doxycycline, suggesting that the newly created single plasmid allowed inducible tyrosinase expression in many different cells lines.
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Robert J. Paproski, Robert J. Paproski, Roger J. Zemp, Roger J. Zemp, } "Inducible expression of photoacoustic reporter gene tyrosinase in cells using a single plasmid", Proc. SPIE 8223, Photons Plus Ultrasound: Imaging and Sensing 2012, 82233Y (23 February 2012); doi: 10.1117/12.909644; https://doi.org/10.1117/12.909644

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