Fig. 5
PS-specific induction of dsRed2. a Schematic diagram depicting the targeting strategy for the construction of the Tps/tb-RED ES cell line. Correctly targeted clones reconstruct a functional Hprt locus and these are therefore resistant to HAT selective medium. Numbers indicate Hprt exons. HAT, hypoxanthine-aminopterin-thymidine. G.O.I. gene of interest; P, promoter; TetOP, tet operator sequences. b Flow cytometry analysis of dsRed2 expression in Tps/tb-RED ES cells cultured in the absence of LIF and the presence of varying concentrations of Dox for four days. E14, E14tg2a; MFI, Mean Fluorescence Intensity. c qPCR expression analysis of indicated markers in sorted dsRed2+ and dsRed2− differentiated Tps/tb-RED ES cells. Results are represented as log10 ratio of expression versus dsRed2− cells. Error bars represent the S.E.M (n = 3). d Immunocytochemical analysis of T(Bra) expression in differentiated Tps/tb-RED ES cells cultured in the presence of Dox. Its correlation with dsRed2 expression as assessed by single cell image analysis is shown in the graph on the right. e dsRed2 expression in the PS of a Dox-treated chimeric E8.0 Tps/tb-RED embryo. The broken line area denotes the node and emergent notochord; the arrow indicates a labelled cell cluster in the node-streak border. (e’-e”) Representative optical confocal image sections of the posterior region of a chimeric E8.0 Tps/tb-RED embryo showing the presence of dsRed2+ cells in the ectoderm (e’) and mesoderm (e”) (arrows). Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). (f-f’”) dsRed2 induction in the PS of Dox-treated E8.5 chimeric C2 (Tps/tb-RED/CAG-GFP) ES derived embryos. GFP expression marks chimeric contribution. NE, neuroectoderm; PXM, paraxial mesoderm. (g-h) dsRed2 expression in the tail bud of Dox-treated E10.5 (g-g”’) and E11.5 (h-h”’) C2 chimeric embryos. In all cases chimeric embryos were treated with Doxycycline for at least 24 h prior to the indicated recovery time point