Strategy and molecular evidence for targeted inactivation of the <i>Abcd2</i> gene.

<p>(A) Structure of the 5′ region of the <i>Abcd2</i> gene, the targeting construct and the disrupted gene. Exons (Ex) 1–3 are depicted as boxes with the protein coding region in gray. After homologous recombination, 1.24 kb of the <i>Abcd2</i> gene, including the transcription and translation start sites and most of exon 1, is replaced by the <i>neomycin resistance</i> gene cassette (Neo) of the targeting construct. Positions of the PCR primers (P1, P2) and the Southern blot probe (****) used in genotyping to detect homologous recombination are indicated. Restriction sites used for cloning or analysis are marked: H, <i>Hind</i>III; N, <i>Nde</i>I; Not, <i>Not</i>I; S, <i>Sca</i>I; Sp, <i>Sph</i>I and X, <i>Xho</i>I. (B) Southern blot analysis of <i>Sca</i>I-digested DNA from <i>Abcd2</i>^−/−, <i>Abcd2</i>^+/+ and <i>Abcd2</i>^+/− mice. Chemiluminescent detection of a digoxygenin-labelled probe from the 5′ flanking DNA verified homologous recombination based on deletion of a <i>Sca</i>I-site in exon 1 of <i>Abcd2</i> resulting in 3.9-kb and 4.4-kb fragments from the wild-type (WT) and knock-out (KO) alleles, respectively. (C) Northern blot analysis of mRNA from brain and skeletal muscle of adult <i>Abcd2</i>^−/− and <i>Abcd2</i>^+/+ mice. A ³²P-labelled <i>Abcd2</i> cDNA probe hybridized to the major 4.2 kb <i>Abcd2</i> mRNA and minor (5.5 and 2.8 kb) variants in both tissues of wild-type mice, but not in <i>Abcd2</i>^−/− mice. As a loading control, the blot was re-probed with β-actin cDNA, which in skeletal muscle detects a shorter, more abundant mRNA than in brain. (D) Reverse transcription-coupled PCR analysis of <i>Abcd2</i> mRNA expression in primary peritoneal macrophages from two <i>Abcd2</i>^+/+ and two <i>Abcd2</i>^−/− mice. <i>Abcd2</i>-specific primers amplified the expected 1,051-bp product in <i>Abcd2</i>^+/+ but not in <i>Abcd2</i>^−/− macrophages. As a positive control, amplification of <i>Iba1</i> cDNA (310-bp fragment) was confirmed in all samples.