Conditional Gene Knockout in Postnatal Mice

Cdh5^CreERT2Krit1^fl/fl mice were generated by breeding Cdh5^CreERT2 and Krit1^fl/fl mice as previously reported.^{14 (link)} All mice were maintained on a mixed 129Sv/C57Bl6 background. At 1-day post-birth, 40 μg of 4-hydroxytamoxifen (H7904, Sigma Aldrich, Milwaukee, WI) were dissolved in 50 μL of warm 5% ethanol/corn oil vehicle (Sigma Aldrich), and were intragastrically injected into mouse pups using a 30-gauge needle. At 10-day post-birth, 1 mg of Avertin (2, 2, 2-tribromoethanol, Sigma Aldrich) was intraperitoneally injected into mouse pups for anesthesia, followed by an intracardiac perfusion of 8 mL of cold phosphate-buffered saline using a 27-gauge needle. The brains were then surgically removed using a standardized procedure and dropped into a 4% paraformaldehyde fixative solution (Sigma Aldrich). The Cdh5^CreERT2 mice were a generous gift from Ralf H. Adams. All procedures were performed in accordance with the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) that approved all animal protocols.