Disruption of the ALAS-E gene in mouse ES cells. (A) Strategy for ALAS-E gene targeting. A phage clone encoding a part of mouse ALAS-E gene was cloned independently in our laboratory and is shown in the upper side of (A) along with the normal mouse ALAS-E gene. A probe used for Southern blotting in (B) is also shown. Maps of the targeting construct and the predicted structure of the targeted ALAS-E allele are shown in the lower lines of (A). (B) Southern blot analysis of ES clones. A linearized construct was electroporated into J1 and CCE cells and 20 mg of genomic DNA was isolated from clones selected in the presence of Neomycin and Gancyclovir. The DNA samples were digested with Pst I or EcoRI and examined by Southern blot analysis. 6.8-kb and 4.6-kb bands detected after digestion withPst I represent the wild-type and the disrupted allele, respectively, while 2.8-kb and 2.1-kb bands detected after digestion with EcoRI represent the wild-type and the disrupted allele, respectively. DNA samples are from wild-type CCE cells (lane 1), ALAS-E(-) J1 cells (lane 2), or ALAS-E(-) CCE cells (lane 3). (C) Heme content in differentiating EBs. Heme content of EBs of day 6, 8, and 10 was determined fluorometrically33 using 1 × 105 cells, which were dissociated from EBs into single cells by incubation in a collagenase solution. Data are the mean of three separate experiments.
