Fig. 5
Assay for analysis of temperature dependence of CREs. (A) Scheme of assay involving an engineered target locus in SR9rg cells for directional RMCE. After insertion of a candidate CRE into an exchange plasmid and cotransfection with a dual integrase expression plasmid (not shown) for production of the PhiC31 and Bxb1 integrases, the chromosomal cassette (with bidirectional blasr and mRuby marker genes) can be replaced with the exchange plasmid cassette where the CRE is in front of the DSCP promoter. After RMCE, CRE activity can drive expression of green fluorescence in the resulting cell population, which can be cultured in aliquots at different temperatures before analysis by flow cytometry. (B) Flow cytometric analysis of enhancer activity of test fragments after RMCE with SR9rg cells. Cartoons of scatter plots with red and green fluorescence intensity along x and y axis depict expected results (from left to right): SR9rg cells before RMCE express mRuby but not mEGFP. RMCE with a test fragment lacking enhancer activity will generate a cell population with neither red nor green fluorescence (grey spot). In contrast, an enhancer fragment will result in a population that expresses only green fluorescence with an intensity depending on enhancer activity. (C) Validation of the SR9rg assay system. Scatter plots after flow cytometric analysis (from left to right): SR9rg cells before and after RMCE with the test fragments 20 × UAS, Rpn13_E1 and ced-6_E. Analyzed windows and percentage of cells therein are indicated. In the rightmost scatter plot, a population of cells expressing both red and green fluorescence is indicated (arrow). (D) Temperature-dependence of CRE activity. Test fragments from genes with temperature-regulated transcript levels (Hsp23) or without (sqh, Rpn13, Karybeta3), as indicated by 3′ RNA-Seq (bar diagrams). After RMCE, aliquots of the cells were shifted to 14, 25 or 30 °C before flow cytometric analysis