FIG. 1.

Rescue strategy of AML1-deficient ES cells by expressing PEBP2αB1 (AML1b) cDNA from a knock-in allele. (A) The AML1 loci of the ES cell lines were disrupted by targeting exon 4, which encodes the middle of the Runt domain; one allele was replaced by a hygromycin resistance cassette [KO(hygr)] and the other by a neomycin resistance cassette [KO(neo)] (37). To rescue the AML1-deficient ES cell phenotype, we designed a replacement-type vector (knock-in vector) which creates an artificial allele [KI(puro)] that expresses the PEBP2αB1 isoform of the AML1 protein in place of the disrupted exon 4 by means of homologous recombination. Clones which had undergone targeted insertion at either of the hygromycin or neomycin alleles were detected by Southern blot analyses with a 5′ outside probe (B) and 3′ outside probe (C), in which the knock-in alleles were detectable as XbaI-restricted fragments of ca. 11 and 7 kb, respectively. Abbreviations: hygr, hygromycin B resistance cassette; neo, neomycin resistance cassette; puro, puromycin resistance cassette; DT-A, diphtheria toxin-A suicide cassette. Arrows above selection cassettes indicate the orientation of the transcription. (D) Expression of the AML1 message in the knock-in ES clones was confirmed by RT-PCR analysis, which amplified the 292-bp fragment of the cDNA with a primer pair corresponding to exons 3 and 4 of the gene locus (top panel). Parallel amplification for the HPRT gene (bottom panel) demonstrates the integrity of the RNA samples obtained from the undifferentiated ES cell clones.