monoclonal control, anti-Fe65 (I12) [19] or non-specific rabbit polyclonal control were added for incubation for 30 min. 20 ul protein A/G was then added for overnight incubation. Superna- tant was removed and the beads were washed 46 with IP buffer. Beads were re-suspended in 16 LDS buffer with 10% BME and 1% NEM and incubated for 5 min at 95uC. Immunoblot analysis was carried out as described above with a-APP IP probed for Fe65 and a-Fe65 IP probed for full length APP (22C11 antibody, Millipore, 1:1500). Pathological evaluation Complete necropsy was performed on all mice and tissues collected were fixed in Tellyesniczky/Fekete fixative (100 ml 70% ethanol, 5 ml 37–40% formalin, 5 ml glacial acetic acid). Appropriate tissues were decalcified using 10% formic acid (Formical-2000H, Decal Chemical Corporation, Tallman, NY) or 3% hydrochloric acid (Cal-ExH, Fisher Scientific, Fairlawn, NJ) for 24 hours. Tissues were paraffin embedded and stained with hematoxylin and eosin (H&E). A board certified veterinary pathologist with no knowledge of the genotypes analyzed the slides. Results Generation of APP Y682G and T668A Mutant Mice The targeting strategy for the generation of the APP KI mice entailed the replacement of APP exon 16 with exon 16 carrying the Y682G or T668A mutation (Fig. 1A). The vector used the floxed PGK-neo selection cassette and contains a 59 homologous region and the negative selection cassette, PGK-dta. The 39 homologous region introduced the T668A or Y682G mutation, BamHI and SmaI sites into the APP mouse gene. The linearized targeting vector was transfected into 129 ES cells. In the presence of the positive selection drug, G418, clones only survived if both the PGK-neo selection cassette was integrated and the PGK-dta cassette was removed by homologous recombination. ES cell clones carrying the targeting vector by random, non-homologous integration, were eliminated due to expression of diphtheria toxin. After selection, ES cell clones carrying the proper homologous recombination and the Y682G or T668A mutant allele were identified by PCR for 39 region (i.e. right Arm: if homologous recombination had occurred these primers would amplify a product of 3.2 Kb). Out of ,600 screened ES clones, we found two targeted clones for T668A and four clones for Y682G mutation (Fig. 1B). Also, PCR amplification and digestion was used to check the proper insertion of the construct in the genomic DNA and the removal of the Neo cassette (not shown). The occurrence of homologous recombination was confirmed by both sequencing and Southern blot analysis (Fig. 1C). DNA derived from individual Y682G or T668A ES clones was digested with BamHI, gel separated, blotted into a nylon membrane and hybridized with the 39probe. The 39 probe hybridizes with a ,7.5 Kb fragment derived from the wild-type locus. Homologous recombination at the 39 homologous region yields a ,6.0 Kb fragment upon BamHI digestion due to the introduction of the BamHI site and the PGK-neo selection cassette. ES clones (T668A or Y682G) carry a wild type allele (7.5 Kb) and a recombined allele (6.0 Kb). The 7.5 Kb and 6.0 Kb bands had a similar intensityproving the ES cells selected were clonal populations. Similar results were obtained when homologous recombination at the 59 site was assessed. Two ES cell clones each for Y682G or T668A (129, agouti coat color), carrying the correct site-specific homologous recombina- tion, were injected into C57BL/6J blastocysts (black coat color). APP Processing and Tyrosine682 The resulting chimeras with a high proportion of agouti coat color (i.e. with a high relative contribution from the injected ES cells) were backcrossed to C57BL/6J mice to obtain heterozygous Y682G/wt, which were identified by PCR and Southern analysis as described above (not shown) using tail DNA. Heterozygous mice were crossed to Meu40-Cre mice to obtain Meu40/APP Y682G/wt or Meu40/APP T668A/wt animals. Cre is a bacteriophage P1-encoded recombinase that catalyzes site-specific recombination between two 34 bp loxP recognition sites, resulting in the excision of the intervening DNA sequences. The resulting mice are named APP Y682G or APP T668A and are abbreviated to APP YG or TA respectively, where appropriate. Reduced b-cleavage and enhanced a-processing of APP Y682G mutant mice Previous studies have shown an important role for Tyr682 in shifting APP toward the amyloidogenic (b-processing) rather than the non-amyloidogenic (a-processing) pathway in vitro [11]. Using the models described above we established whether this is true in vivo. Rates of a and b processing are reflected by the relative abundance of the products of these two pathways, sAPP-a and sAPP-b, respectively. Using immunoblot analysis an approximate- ly 15-fold increase in sAPP-a in conjunction with a 3.5 fold decrease in sAPP-b was detected in brain tissue from APPYG/YG mice compared to APPwt/wt controls (Fig. 2A & 2B). Importantly, no differences in sAPP-a and sAPP-b between APPTA/TA, APPTA/ wt and APPwt/wt mice (Fig. 2C) was detected thus demonstrating the highly specific role of Tyr682 in modulating entry of APP into the amyloidogenic pathway in vivo. An analysis of APP COOH-terminal fragments (Fig. 2D) shows that C83, which is formed in conjunction with sAPP-a, is greatly increased in APPYG/YG mice over APPwt/wt control, consistent with an increase in non-amyloidogenic processing. The level of C99, which is formed in conjunction with sAPP-b, does not change appreciably. Interestingly, the level of the phosphorylated APP COOH-terminal fragments, p-C99, p-C89 and p-C83, were unchanged in APPYG/YG but, consistent with observations by Sano, Y. et al. [20], were below detectable levels in APPTA/TA mice. This indicates that the steady-state phosphorylation of APP is predominantly located on Thr668 in the brain and that phosphorylation of Tyr682 is highly regulated (perhaps by signaling mechanisms) and may have a short half-life. Also, it shows that the Y to G mutation has not grossly altered the structure of the APP intracellular domain that since the mutant APPYG protein still undergoes other phosphorylation events. Other products of the amyloidogenic pathway are Ab40 & 42. Only Ab40 was detectable and showed a significant 25% decrease in APPYG/YG mice compared to APPwt/wt controls (Fig. 2E), which was also consistent with a decreased b-processing of APP. The change was specific for the Y682G mutation since Ab40 levels were not changed in the APPTA/TA mice, again in agreement with previous findings [20]. Total APP levels show a 64% increase in APPYG/YG over controls, after normalization with Tubulin (Fig. 2A & B). Whether this indicates an increased total half-life of APP Y682G as compared to WT APP remains to be determined. It is also possible that manipulation of the APP gene locus may have altered the transcription/splicing of the mutant allele. To test for this, we have now performed real-time RT-PCR and the data demonstrate that expression levels of APP mRNA are not affected by the genetic strategy used to do the KI (Fig. 3A). Thus any alterations in APP protein and in its metabolites almost certainly reflect an effect of the YG mutation on the fate of the APP protein, since it is very unlikely that the point mutation will affect the translational efficiency of the KI mRNA. Moreover, this increase in total APP is PLoS ONE | www.plosone.org 4 November 2010 | Volume 5 | Issue 11 | e15503