Development and analysis of Cdh5^−/−GFP^+/+: Cdh5^+/+GFP^−/− chimeric mice embryos. (A) Schematic representation of the Cdh5 chimeric mouse development and analysis. Identification of Cdh5^−/−GFP^+/+ mES cells after electroporation of Cdh5 CRISPRs targeting exons 1 to 12 of the Cdh5 gene followed by karyotyping to exclude aneuploidy. Injection of Cdh5^−/−GFP⁺ mES cells into WT mouse embryo of foster mothers and harvesting chimeric AGM at E10.5 for immunohistochemistry (GFP, Runx1, DAPI) and at E11.5 for cd41/c-Kit/cd45.2 expression, CFU analysis, transplant into sublethally irradiated mice as well as at E13.5 fetal liver for Lineage⁻Sca1⁺c-Kit⁺ expression, CFU analysis, and reconstitution capacity of fetal liver transplants. (B) Western analysis for Cdh5 and Hsp60 protein expression in E13.5 Cdh5^−/−GFP⁺:Cdh5^+/+GFP⁻-fetal liver–derived GFP⁻ and GFP⁺ cells. To demonstrate the absence of Cdh5 at protein level, we sorted GFP⁺ and GFP⁻ cells from fetal liver of E13.5 Cdh5^−/−GFP^+/+:Cdh5^+/+GFP^−/− chimeric mice embryos. Our western blot data confirmed the absence of Cdh5 protein in GFP⁺ cells. (C) The percentage of GFP chimerism in 11 E11.5 AGM, 6 E13.5 fetal liver, and 3 live pups. (D) Immunostaining of GFP (green), Runx1 (red), and DAPI (violet) in transverse sections of E10.5 AGM region, indicating that Cdh5^−/− GFP⁺ cells produced Runx1⁺ GFP⁺ hematopoietic clusters in the ventral wall of the E10.5 dorsal aorta. Magnification, 40×.