Fig 1.

Expression of the M_r 175,000 Dnmt1o protein from the Dnmt1 1s promoter by mutating the translation initiation codon in exon 1s. (A) The transcripts for Dnmt1o and Dnmt1 (called Dnmt1o and Dnmt1, respectively) are normally produced from different promoters associated with exon 1o and exon 1s, respectively. The Dnmt1o first exon (exon 1o) and the Dnmt1 first exon (exon 1s) are both spliced into the common exon 2 (4). Exon 1p is an alternative first exon whose associated promoter is active in spermatocytes (4). The linear targeting construct contains a mutated ATG initiation codon in exon 1s and a loxP-flanked cassette of HSV-tk and Pgk1-neo^r genes at a BamHI site in the intron between exon 1p and exon 2. The loxP sites are filled triangles. After homologous recombination between the targeting construct and a WT Dnmt1 allele, and removal of the antibiotic-resistance genes by Cre-mediated excision, the mutant Dnmt1^V allele is produced. The Dnmt1 protein is synthesized from 1s transcripts of the Dnmt1 allele, and the Dnmt1o protein is synthesized from 1s transcripts of the Dnmt1^V allele. B, BamHI; RV, EcoRV. (B) Southern blot confirmation of homologous recombination at the Dnmt1 locus. DNA was digested with EcoRV, and hybridization was performed with Probe A (A). v = Dnmt1^V allele; + or wt = WT Dnmt1 allele or locus. (C) Effect of Cre-mediated deletion of HSV-tk/Pgk1-neo^r from targeted allele on protein expression in ES cells. Dnmt1o and Dnmt1 proteins were detected with an immunoblot assay by using the UPTC21 Ab (9). −Cre, before Cre-mediated excision; +Cre, after Cre-mediated excision. (D) Genotyping of Dnmt1 and Dnmt1^V alleles by using primers flanking exon 1s followed by EcoRV digestion of PCR product. EcoRV digests Dnmt1^V allele to yield 220- and 80-bp products.