FIGURE 1.

Analysis of mouse Atat1 by gene targeting. A, strategy for generation of the Atat1-null allele. Pink boxes denote the 5′- and 3′-untranslated sequences, and solid boxes indicate exons of the Atat1 gene located at the mouse chromosome 17qB1 (UCSC Genome Browser). A Bac targeting vector was utilized to replace the 9.2-kb genomic sequence of Atat1, spanning the entire coding region, with the promoterless lacZ coding sequence and a ubiquitin C (UBC) promoter-neomycin selection cassette flanked by two LoxP sites. SA, splicing acceptor; pA, polyadenylation signal. Rough positions of the PCR genotyping primers are indicated with tiny bars; F1 and R1 are for amplifying a 321-bp fragment from wild-type allele, and InF and InR are used to amplify a 210-bp fragment from the knock-out allele. B, PCR genotyping with the indicated primer pairs. The asterisk denotes nonspecific bands. M indicates the 100-bp DNA ladder as the molecular size marker (lane 1). C, adult Atat1^−/− mice are indistinguishable from wild-type littermates by gross examination. D, RT-PCR analysis of Atat1 expression in tissues from the wild-type and homozygous knock-out mice. Atat1 mRNA was detected in the indicated tissues from the wild-type but not Atat1^−/− mouse. Gapdh, internal control. E, RT-PCR analysis of lacZ mRNA in various tissues from the wild-type and knock-out mice. The cDNA templates were the same as those used in D, with PCR primers Lac-InF/-InR (A). M, 100-bp DNA ladder (lane 1).