FIGURE 1.

Genomic editing of APP gene locus. TALEN pairs were designed to target and induce a double strand break (DSB) in the first exon upstream of the normal APP translation initiation codon (APP ATG). The DSB was repaired by homologous recombination in the presence of plasmids containing the coding sequence for either Aβ40 or Aβ42 fused in frame with a rat preproenkephalin secretory signal sequence (SS) and followed by a polyA tail. Repair plasmids additionally included a PGK puromycin drug selection gene (Puro) and were flanked by left and right homology arms homologous to APP flanking sequences (HAL, HAR). Cassette insertions were confirmed by genomic PCR using specific primers in either the HAL (5′) or the HAR (3′) and a site in the insertion cassette. This editing strategy simultaneously inactivates one APP allele and replaces it with a cassette that directly expresses a secretory form of either Aβ40 or Aβ42 under normal APP regulatory control. The specific sequences and other details are included in the Supplementary Methods and Data.