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Figures
- Fig. 1. Hprt targeting strategy and cardiac enhancer activity in beating cardiomyocytes.
(A) The Hprt locus in mouse HM-1 ES cells lacks the Hprt promoter, exon 1 and exon 2. The Hprt targeting vector contains the enhancer of interest, the Hsp68 minimal promoter and Venus fluorescent reporter gene upstream of the human HPRT promoter, human exon 1 and mouse exon 2 and these are all flanked by Hprt locus homology arms. Homologous recombination of this targeting construct in HM-1 ES cells results in reconstitution of the Hprt locus and insertion of the enhancer/Hsp68/Venus reporter upstream of the promoter. Use of Hprt substrate analogues to select for HM-1 with deficient or reconstituted Hprt provides a stringent selection method for selection of correctly targeted clones. (B) Bright field (above) and fluorescent (below) images of representative beating embryoid bodies (EBs) at day 14, from HM-1, Hsp68/Venus control, mm75/Hsp68/Venus and mm77/Hsp68/Venus clones. (C) Average percentage of spontaneously beating EBs that are YFP positive at day 14 for the above clones, average of two independent differentiation experiments.
- Fig. 2. Gfi1-35 activity marks haematopoietic-fated cells during embryoid body differentiation.
(A) Fluorescent images of day 3–6 EBs from the representative clones of HM-1 (top), Hsp/Venus control (middle) and Gfi1-35/Hsp/Venus (bottom) lines. (B) Representative flow cytometry plots of Venus-YFP vs side scatter (SSC) for HM-1 (top), Hsp/Venus control (middle) and Gfi1-35/Hsp/Venus (bottom) day 4 EB cells with the YFP positive gate, and its percentage of the live cell population, shown in red. (C) Percentage of the YFP positive population (gating shown in B) from day 3–7 for HM-1 (blue), two representative Hsp/Venus control clones (aqua and purple) and two representative Gfi1-35/Hsp/Venus (red and orange) clones, showing average of three independent differentiation experiments ± standard deviation. (D) Representative flow cytometry plots showing distribution of Gfi1-35/Hsp/Venus day 4 EB cells in Flk1/CD41 quadrants for all live cells (left plot) and YFP positive cells only (right plot), with the percentage of cells in each quadrant shown in red. (E) Percentage of YFP positive cells in each of the Flk1/CD41 quadrants of (D) for Gfi1-35/Hsp/Venus (clone 1 in red, clone 2 in orange). Average of three independent differentiation experiments ± standard deviation. (F) As in (D), but for Flk1/CD140a quadrants. (G) As for (E), but for Flk1/CD140a quadrants in (F).
- Fig. 3. Scl+19 activity marks haematopoietic cells and cardiac mesoderm in differentiating embryoid bodies.
(A) Fluorescent images of day 3–6 EBs from a representative Scl+19/Hsp/Venus clone. (B) Representative flow cytometry plots of Venus-YFP vs side scatter (SSC) of day 4 EBs for Scl+19/Hsp/Venus line with the YFP positive gate, and its percentage of the live cell population, shown in red. (C) Percentage of the YFP+ population (gating shown in B) from day 3–7 for HM-1 (blue), two representative Hsp/Venus control clones (aqua and purple) and two representative Scl+19/Hsp/Venus clones (red and orange), showing the average of three independent differentiation experiments ± standard deviation. (D) Representative flow cytometry plots showing distribution of Scl+19/Hsp/Venus day 4 EB cells in Flk1/CD41 quadrants for all live cells (left plot) and YFP positive cells only (right plot), with the percentage of cells in each quadrant shown in red. (E) Percentage of YFP positive cells in each of the Flk1/CD41 quadrants of (D) for Scl+19/Hsp/Venus (clone 1 in red, clone 2 in orange). Average of three independent differentiation experiments ± standard deviation. (F) As in (D), but for Flk1/CD140a quadrants. (G) As for (E), but for Flk1/CD140a quadrants in (F).
- Fig. 4. Dissection of the Scl+19 enhancer by motif mutation.
(A) Sequence conservation of the Scl+19 core region in mouse, human, dog and opossum, with Ets motifs highlighted in yellow and the Gata motif highlighted in green. (B) Representative flow cytometry plots of Venus-YFP vs side scatter (SSC) of day 4 EBs for Scl+19/Hsp/Venus and Scl+19 mutant clones with the YFP positive gate, its percentage of the live cell population and mean fluorescence intensity (MFI), shown in red.
- Fig. 5. Gata2 is not required for endothelial activity of the Scl+19 enhancer in E9.5 transgenic mouse embryos.
(A) Gata2+/− mice were crossed with mice carrying an Scl+19 reporter (Scl-3′enh/SV/lacZ) to produce Gata2+/− Scl-3′enh/SV/lacZ mice. These were re-crossed with Gata2+/− mice, from which E9.5 embryos were collected and genotyped for Gata2 and Scl-3′enh/SV/lacZ phenotypes. (B) Representative whole mount images of E9.5 mouse embryos, genotypes shown above, stained for lacZ expression using Xgal.
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