PMC:1283364 / 6438-9425 JSONTXT

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This ligation generated a 9.3-kb vector encoding the tetracycline promoter from pTetSplice with two exons, one intron, and the original 3′ UTR of the moPrP.XhoI vector, all carried in the pBluescript cloning vector.\nWe incorporated the Swedish (KM570/571NL) and Indiana (V617F) mutations into the mo/huAPP695 cDNA (in BS-KS) by PCR using a four-primer strategy: first, two partially overlapping products were generated in separate reactions using primers that encode the desired mutations (Swedish forward: GGA GAT CTC TGA AGT GAA TCT GGA TGC AGA ATT CCG/Indiana reverse: GGG TGA TGA AAA TCA CGG TTG C; Indiana forward: CAA CCG TGA TTT TCA TCA CCC TGG/M13 reverse). The two PCR products were ligated, digested with BglII and ApaI and cloned back into the original mo/huAPP695-BS-KS vector. Finally, the new APP695swe/ind was subcloned into the XhoI site of the moPrP-tetP vector from above to complete the construct.\n\nPronuclear Injection, Screening of Founders, and Maintenance of the Lines\nThe moPrP-tetP-mo/huAPP695swe/ind vector was linearized and the pBluescript domain excised by digestion with NotI. The purified vector was injected into the pronucleus of fertilized eggs from C57BL/6J × C3HeJ F1 matings. Founder animals were screened for the presence of the transgene by three-way PCR using the S36 and PrP-S/PrP-AS primers described below. Transgene-positive founders were bred to animals expressing the tetracycline transactivator (tTA) under control of the calcium-calmodulin kinase IIα (CaMKIIα) promoter obtained from Jackson Laboratory [16] (Bar Harbor, Maine, United States; stock # 3010; B6;CBA-TgN[Camk2a-tTA]1Mmay). The colony was thereafter maintained by crossing single transgenic tTA and APP offspring for each of the four APP lines. All mice were provided fresh food and water ad libitum. Animal protocols were approved by both the Johns Hopkins University and the California Institute of Technology Institutional Animal Care and Use Committees.\n\nDoxycycline Administration\nDoxycycline (dox) was administered through commercially available dox-containing chow (BioServ, Frenchtown, New Jersey, United States). The chow contained 200 mg/kg of antibiotic; based on estimated consumption of 5 g per mouse per day, the expected dose to each animal was 1 mg dox per day. 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