Targeted disruption of the murine <i>Zfyve26</i> gene.

<p>(A) Genomic structure of the <i>Zfyve26 gene</i> (top) and the targeted <i>Zfyve26</i> locus (middle). The dotted line indicates the extent of the targeting construct. A neomycin cassette (Neo) flanked by frt sites (black boxes) and a loxP-site (black triangle) was inserted into intron 15. A second loxP-site together with a <i>Bam</i>HI site was introduced into intron 14. Correctly targeted ES cell clones were selected for the generation of chimeric mice. <i>Zfyve26</i> knockout mice were established by breeding chimeric mice to a cre-deleter mouse strain to obtain constitutive <i>Zfyve26</i> knockout mice. (B) Northern blot analysis of total brain RNA from wild-type (WT), <i>Zfyve26</i> heterozygous (HET), and <i>Zfyve26</i> knockout (KO) animals. <i>Gapdh</i> served as loading control. (C,D) Western blot analysis with affinity purified antibodies against the N-terminus (C) or the C-terminus (D) of Zfyve26 detected a 285 kD Zfyve26 polypeptide in brain extracts from wild-type but not from knockout mice. Calnexin served as a loading control. (E) Spatacsin levels, an interaction partner of Zfyve26, are reduced in brain lysates of <i>Zfyve26</i> knockout mice. Calnexin was used as a loading control. (F) <i>Spatacsin</i> is not regulated on the transcriptional level in <i>Zfyve26</i> knockout mice. <i>Gapdh</i> was used as a loading control.