Fig. 6: CDH5-MAPK and Tie2.IkB-SS mice demonstrate endothelial-specific expression of transgenes.

a Schematic describing breeding strategy for generation of CDH5-MAPK mice. b Schematic describing Tamoxifen regimen before experimental analysis. Representative flow cytometry contour plots demonstrating GFP expression in whole-bone-marrow cells of CDH5-MAPK mice. Numbers indicate average frequency of cells in the indicated quadrants as the percentage of total BM cells ± SEM (n = 4–5 mice/cohort). Note that GFP expression is detected exclusively in cells within the CD45− fraction of whole-bone-marrow cells and demonstrate surface expression of endothelial markers. c Analysis of phospho-ERK1/2 expression by flow cytometry confirms in vivo activation of the MAPK pathway in BMECs of CDH5-MAPK mice following Tamoxifen administration (n = 3 mice/cohort). d GFP+ BMECs as compared to GFP− BMECs within CDH5-MAPK mice demonstrate increased phospho-ERK1/2 expression by flow cytometry, confirming the fidelity of GFP reporter for tracking cre-mediated recombination in vivo (n = 3 mice/cohort). e Endothelial cells within the BM of CDH5-MAPK mice (defined as CD45− Ter119− CD31+ VEcadherin+) demonstrate cre-mediated recombination whereas stromal cells (defined as CD45− Ter119− CD31− VEcadherin−) do not (n = 4 mice/cohort). f Schematic describing the Tie2.IkB-SS mouse model. g Agarose gel electrophoresis image of RT-PCR amplicons for the indicated genes using RNA isolated from FACS-sorted endothelial cells and CD45+ hematopoietic cells in the indicated genotypes (n = 3 mice/cohort). Note that IkB-SS transgene is expressed in endothelial cells and shows no detectable expression in hematopoietic cells. Also note the expression of cre transgene in endothelial cells of CDH5-MAPK mice and no detectable expression in hematopoietic cells. NTC denotes “No Template Control”. Sort purity was confirmed using expression of Cdh5 (for endothelial cells) and Ptprc (for hematopoietic cells). Error bars represent sample mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t-test. *P ≤ 0.05; **P < 0.01.