Regulation of transgene expression in ERT2-Gal4 and UAS transgenic lines using tamoxifen. (a) Schematic diagram of crosses from ERT2-Gal4 and UAS transgenic zebrafish. The ERT2:Gal4 transgenes carry a cryaa:RFP cassette therefore transgenic fish can be identified by red fluorescence in the lens. The UAS transgenes carry a myl7:EGFP cassette which drives in GFP expression in the heart. This allows identification of transgenic carriers regardless of UAS expression. From crosses of UAS and Gal4 transgenic fish, 25% of offspring will inherit both transgenes. (b) Tamoxifen induces UAS-driven transgene expression in double transgenic larvae. Offspring of ubb:ERT2-Gal4 and UAS:mRFP-GFP-lc3 were either treated with DMSO or 1 µM tamoxifen. In control (DMSO) conditions, ERT2-Gal4 is inactive as it is retained in the cytoplasm. Therefore, there is no UAS-driven expression in double transgenic zebrafish (red fluorescence in the lens and green fluorescence in the heart demonstrates that the larva carries both transgenes). Upon tamoxifen treatment, ERT2-Gal4 translocates to the nucleus, binds to UAS and activates transgene expression and both green and red fluorescence are seen throughout the body. (c) Tamoxifen treatment does not affect autophagic flux. Lc3-II levels were measured in double transgenic larvae carrying ubb:ERT2-Gal4 and UAS:EGFP transgenes. In control conditions, GFP is not expressed. Treatment with 1 µM tamoxifen induces strong GFP expression but has no effect on Lc3-II levels in either basal or NH4Cl-treated conditions. A nonspecific band (red arrowhead) is present in all lanes and runs just below the level of GFP. This band is obscured by the GFP-positive band in lanes 2 and 4.
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