FIG. 3.

Targeted disruption of the H1e gene in mouse ES cells and mice. (A) Homologous recombination strategy in ES cells. The H1e targeting vector (top) was constructed by removing a 720-bp MscI fragment from a 6.5-kb EcoRI H1e genomic DNA fragment cloned in pGEM-3Z (Promega), containing the H1e coding region (open box), and inserting by blunt-end ligation a 1.8-kb ApaI/HindIII fragment (shaded box) from pPGK-NEO. A 1.8-kb SalI/XhoI fragment from pMCI-TK was inserted into the SalI site at the 5′ end of the gene. A homologous recombination event (X's) between the targeting vector and the endogenous H1e locus (middle) results in production of a modified H1e locus (bottom), in which a 720-bp fragment, including the entire H1e coding sequence, along with 49 bp of the 5′ noncoding sequence and 11 bp of the 3′ noncoding sequence, is removed. (B) Identification and confirmation of ES cell clones containing the modified H1e allele. ES cell DNA (10 μg) was digested with EcoRI, followed by Southern blot analysis using the outside probe shown in panel A. The expected positions of the hybridizing fragments from the unmodified (wild-type) and modified H1c loci and their respective sizes are indicated. Clones analyzed in lanes 1, 4, 5, 8, 9, 11, 13, 15, 17, 18, and 23 underwent a homologous recombination events. (C) Genotype analysis of offspring from parents heterozygous for the modified H1e allele. Siblings that were heterozygous for the modified H1e allele were bred, and 15 μg of tail DNA from offspring was digested with EcoRI, blotted, and hybridized with the inside probe shown in panel A. The deduced genotype of each animal is indicated above each lane. The wild-type and modified loci and their corresponding sizes are indicated.