Generation and hematopoietic analysis of embryos chimeric for Notch1-deficient (Notch1^Δ1 allele) or wild-type (control) cells marked by ROSA26. (A) Schematic representation of generation of chimeric embryos. Primary Notch1^–/– or wild-type ES cell lines containing the ROSA26 gene were independently derived from blastocysts. These ES lines were injected into wild-type blastocysts to obtain chimeric embryos. At various stages of development (Table 1), the yolk sac (YS), a portion of fetal liver (FL), or bone marrow (BM) was dissected out and dispersed cells were plated for hematopoietic CFU activity and the remaining embryo (or dissected organs) was stained with X-gal. Only those embryos that contained widespread, substantial contribution of LacZ⁺ cells by gross visualization of stained tissues were included in the hematopoietic analysis. Hematopoietic colonies were stained with X-gal and CFUs scored in order to determine relative percent contribution of ES-derived (LacZ⁺) and blastocyst-derived (LacZ^–) cells. (B) A representative LacZ⁺ colony from Notch1-deficient (ES derived) cells from a chimeric embryo and LacZ^– colony from Notch1^+/+ (embryo derived) cells from a chimeric embryo. Images were visualized using Olympus 60 ×/1.4 oil objective lenses. (C) DNA samples from single LacZ^– (embryo-derived Notch1^+/+) or LacZ⁺ (ES-derived Notch1^–/–) colonies were isolated and used for PCR to genotype colonies.