brain [10]. This residue forms an essential part of the evolutionarily conserved, canonical endocytic Y682ENPTY687 motif. Compromised endocytosis of APP is shown to significantly decrease amyloidogenic processing and Ab secretion [11]. This motif is also a docking site for cytosolic proteins, such as Fe65, that regulate APP metabolism and signaling [12]. Phosphorylation of Tyr682 promotes interaction of Src-Homology 2 domain (SH2) while it reduces interaction with a subset of proteins containing a Phospho-Tyrosine-Binding (PTB) domain in vitro [13,14]. The potential role of Tyr682 phosphorylation state as a ‘‘biochemical switch’’ to change the molecular composition of APP complexes is an intriguing possibility. In the AD brain, a possible pathological role for augmented APP phosphorylation on Tyr682 needs further exploration. To begin to specifically dissect the functional role of APP intracellular domain in vivo we have generated APP KI mice with a mutation in amino acid Tyr682. Here we describe the generation and initial characterization of APP KI mouse with mutation of Tyr682mutation of Tyr682. Materials and Methods Ethics Statement Mice were handled according to the Ethical Guidelines for Treatment of Laboratory Animals of Albert Einstein College of Medicine. The procedures were described and approved in animal protocol number 20040707 Generation of APP Y682G and T668A mutant Mice A 7.0-Kb genomic fragment containing exon 16 from C57BL/6 BAC DNA (RP23-99P18) was amplified by PCR with the following primers: Fwd: 59-aaaaGGTACCagtatctcttgtcctcacaatg-39; Rev: 59-aaaaCCGCGGtaggtcccaaggcta-39. This fragment flanked by KpnI and SacII sites was cloned into pBS (pBS-EX16), and used as a template for subsequent cloning. Two nucleotide mutations were introduced into pBS-EX16 vector by site directed mutagenesis PCR. Firstly a SmaI/XmaI restriction site (CCC GGA R CCC GGG) was created right in front of exon 16 by using the following primers: Fwd: 59-ctattttaaacccggatctctgtacctgctttc-39; Rev: 59-gaaagcaggtacagagatccgggtttaaaatag-39. This new restriction site was used to verify the targeted clone. Furthermore the nucleotide change from either ACC to GCC or TAT to GGA in exon 16 generated the corresponding amino acid mutation T668A or Y682G, respectively. The following primers were used for this mutation: for T66A mutation, Fwd, 59- TCGACGCCGCCGTGGCCCCAGAGGAGCGC- CATCT -39; Rev, 59- AGATGGCGCTCCTCTGGGGCCACGGCGGC- GTCGA -39; And for Y682G mutation, Fwd, 59-tgcagcagaacggaggagagaatccaact -39; Rev, 59- agttggattctctcctccgttctgctgca-39. The 1.3 Kb Hind III-Sal I left arm and 2.4 Kb NotI-SacII right arm were amplified from above mutated pBS-Ex16 by using the following primers. For right arm: Fwd, 59- aaaaaaagcttcaatggccatgaagga - 39; Rev, 59-aaaaagtcgaccaggtatctgctgccat-39; For left arm: Fwd, 59- aaaaagcggccgcggccccacaaagcggagt -39; Rev, 59- aaaaaccgcggtggcgcatgctgcag- 39. The left arm contains SmaI/XmaI and either T668A or Y682G mutations. Subsequently, the left arm and right arm were inserted APP Processing and Tyrosine682 into a Soriano PGK-Neo-dTA vector. The resulting construct was thus: --- dTA cassette-Left Arm-LoxP1-PGK-Neomycin cassette- LoxP2-Right Arm --- The resulting construct was linearized with KpnI and purified prior to injection in ES cells strain 129 by electroporation. ES culture was performed on feeder layer, and further electroporation and handling was also performed according to the methodology employed at Dept of Cell Biology, Albert Einstein College of Medicine, and according to Wakayama et al. In particular, after electroporation, ES cells were re-plated in 55 cm2 dishes and grown until visible clones appeared. Clones were then picked and transferred to 96 well plates in triplicates. Triplicates were either screened by PCR or frozen for subsequent use and further analysis. Homologous recombinants were selected with G418 (200 mg/ ml) and dTA exclusion. Injection of the two Y682G mutant targeted ES cell clones into C57BL/6J blastocysts was performed at the Albert Einstein College of Medicine gene-targeting facility, according to the facility protocol. PCR Analysis The PCR screening was performed using the Expand Long Template PCR System (Roche-applied-Science) with Betaine, according to the manufacturer instructions. PCR analysis of recombinant ES cells and mice was conducted with the following primers and digestion strategies to identify the correct recombi- nant clones and strains: ES cells: Left arm: Genomic primer: 59-CAGAAGGAAATGTCCCAGGA-39 Neo cassette primer: 59-CTTCTAGTTGCCAGCCATCTG- 39 Product: 1687 bp Right arm: Genomic primer: 59-GGATCTCACCCTGTTTTCCA-39 Neo cassette primer: 59- TGCACGAGACTAGTGAGAC- GTG-39 Product: 3306 bp Amplification of the right arm and digestion: Genomic upstream primer: 59-CTACAGAGATAAATGTAC- TTCG-39 Genomic downstream primer: 59-GGATCTCACCCTGT- TTTCCA-39 Product: 3200 bp = wild type without SmaI/XmaI restriction siteR3200 bp Product: 3200 bp = mutant alle with SmaI/XmaI restriction site; SmaI digestionR2800 bp +400 bp Mice genotyping: Fw primer: 59-ATGGCACCACCCACAATAGG-39 Rev primer: 59-CCTAGCAACTGGTAACAGTGC-39 Product: 2027 bp with Neo cassette Product: 332 bp without Neo cassette Product: 194 bp wild type PCR products were digested to ascertain that the targeted sequence was correctly inserted in the genomic DNA. Southern Blot Analysis Twenty mg of genomic DNA was digested with BamHI overnight, run on a 1% TAE agarose gel and transferred on a Hybond-N+ membrane (Amersham). The probe was prepared by PCR from a BAC clone (RP23- 99P18) with the following primers: -Left arm: PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e15503