Generation of Thr⁶⁶⁸Ala Knock-in Mutant Mice.

<div><p>(A) The targeting vector (a1), a partial map of the APP gene (a2), the resultant targeted allele (a3), and the knock-in allele after Cre recombination (a4) are illustrated.</p> <p>Filled boxes denote coding sequences of exons 17 and 18.</p> <p>Shaded parts in exon18 correspond to the 3′ non-coding region.</p> <p>A substitution is represented by a dot in exon 18.</p> <p>B, <i>BamHI</i>; X, <i>XhoI</i>.</p> <p>Probes for Southern blot analysis for screening of targeted ES clones are indicated with small bars in (a2–4).</p> <p>PCR primers (E1 and E2) for genotyping of mice are indicated with small arrows in (a2 and a4).</p> <p>(B) Verified sequences from wild-type (T/T), and Thr⁶⁶⁸Ala mutation homozygotes (A/A).</p> <p>The Thr⁶⁶⁸ flanking genomic region was amplified using mouse tail DNA as the template by PCR and sequenced.</p> <p>(C) Southern blot analysis for targeted ES cells and PCR analysis for genotyping wild-type and knock-in mouse lines.</p> <p>DNA from G418-selected ES cells was digested with <i>XhoI</i> and analyzed by Southern blotting with a 3′ external probe.</p> <p>The 13-kb and 15-kb fragments represent wild-type and targeted alleles, respectively. PCR fragments of 200-bp and 298-bp represent wild-type and knock-in alleles, respectively.</p> <p>(D) Immunoblot analysis of mouse whole brain.</p> <p>APP was immunoprecipitated with anti-pan APP polyclonal antibody followed by immunoblotting with anti-pan APP antibody UT-421 or anti-phospho-Thr⁶⁶⁸-specific antibody UT-33 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000051#pone.0000051-Iijima1" target="_blank">[7]</a>.</p> <p>APP from homozygotes has no immunoreactivity with UT-33 although the APP expression levels detected by UT-421 were indistinguishable from those of wild-type mice.</p> <p>Mature APP (mAPP; <i>N</i>- and <i>O</i>-glycosylated form), immature APP (imAPP; <i>N</i>-glycosylated form), and phosphorylated APP (pAPP) are indicated with arrows.</p>