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. 2015 Apr 22;4(4):e001972. doi: 10.1161/JAHA.115.001972

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© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

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Figure 1. — Validation of Brg1 excision using vascular‐specific cVECad‐Cre and Cdh5(PAC)‐Cre^ERT² transgenes. A, Immunohistochemistry of E15.5 jugular veins (JV; top images) from littermate control (Brg1^fl/fl) and mutant (Brg1^fl/fl;cVECad‐Cre) embryos immunostained for BRG1 (brown) and counterstained with hematoxylin (blue). Bottom images show BRG1 expression in blood vessels of control (Brg1^fl/fl) and mutant (Brg1^fl/fl;cVECad‐Cre) adult LLC tumors. Asterisk (*) highlights endothelial cells with reduced BRG1 expression. Scale bar=50 μm. B, Tissue samples of heart, quadriceps, and retina from ROSA26R^L^acZ control animals or ROSA26R^L^acZ;Cdh5(PAC)‐Cre^ERT² mutants were whole‐mount X‐gal stained (blue) to detect Cre activity and sectioned. Heart and quadriceps tissues were counterstained with nuclear fast red. Scale bar=50 μm. C, BRG1 expression (brown) in blood vessels of control (Brg1^fl/fl) and mutant (Brg1^flfl;Cdh5(PAC)‐Cre^ERT²) adult LLC tumors. Sections were counterstained with hematoxylin (blue). Arrows denote endothelial cells with BRG1 expression; asterisks highlight endothelial cells with reduced BRG1 expression. Scale bar=50 μm. D, PCR genotyping of DNA isolated from tail clips of a litter of P7 pups (lanes 1 to 11) generated from a cross between a Brg1^fl/fl female and a Brg1^fl/fl;Cdh5(PAC)‐Cre^ERT² male. Top image shows PCR products using primers to detect Cdh5(PAC)‐Cre^ERT², and bottom image shows PCR products using primers to detect a Brg1^fl/fl excision event (Brg1Δ, top band) and an internal control gene to validate sample quality (Tcrd gene; bottom band). Tissue from a Brg1^fl/+;Cdh5(PAC)‐Cre^ERT² animal and sample lysis buffer are included as positive and negative controls, respectively. E, PCR on DNA isolated from various tissues of a 14‐week‐old Brg1^fl/fl;Cdh5(PAC)‐Cre^ERT² female. Top image shows PCR products using primers to detect Cdh5(PAC)‐Cre^ERT², and bottom image shows PCR products using primers detecting a Brg1^fl/fl excision event (Brg1Δ, top band) and an internal control gene to validate sample quality (Tcrd gene; bottom band). Tissue was collected from ear, heart, quadriceps, and whole eye as indicated. Tissue from a Brg1^fl/+;Cdh5(PAC)‐Cre^ERT² animal and sample lysis buffer are included as positive and negative controls, respectively. Brg1 indicates Brahma‐related gene 1; LLC, Lewis lung carcinoma; PCR, polymerase chain reaction; Tcrd, T‐cell receptor delta chain gene.