Generation of Ung^–/– mice and the resulting DNA, RNA, and activity analysis. (A) Targeting strategy for inactivation of the ung gene. Lines (from top to bottom) show the map of the ung genomic locus, which consists of six exons (and exon 1a), the structure of the targeting construct (neo construct), and the resulting mutant (neo) allele after homologous recombination. Homologous recombination leads to the deletion of both mitochondrial (UNG1, exon 1a) and nuclear (UNG2) isoforms of uracil DNA glycosylase. Relevant Southern blot analysis was performed with the indicated 3′ outside probe. EcoRI digestion results in an 8.2-kb fragment in wild-type and a 5.2-kb fragment in homologus recombinations. (B) Exemplary Southern blot analysis of EcoRI-digested DNA from ES cells and mouse tissue (tails). neo, mutant. (C) Activity assays that measured the ability of Ung^+/+ and Ung^–/– ES cell lysates to excise uracil from an end-labeled oligonucleotide substrate demonstrated no detectable enzymatic activity in the knockout cells (independent experiments). (D) RT-PCR in which Ung^+/+ and Ung^–/– ES cells were used, demonstrated residual transcription of exons 1a and 6 in the recombined ung genomic locus. The promoter region for exon 1a was left intact by the knockout strategy and allowed residual transcription.