(A) Rat PAC clone and vector map (27, 52). (B) Strategy for targeted PAC modification according to ref. 28. This panel was adapted from ref. 28 with permission of Nature biotechnology. The modification cassette, inserted in the shuttle vector, contains flanking rat Notch3 genomic sequence of exons 3 and 6 on both sides of the engineered CADASIL mutation in exon 4. Two PAC modification steps are illustrated: co-integration of the shuttle vector into the PAC (1st and 2nd) and the resolution of the co-integrant by a second homologous recombination event to eliminate the shuttle vector and other exogenous sequences leaving the modified PAC carrying a point mutation. (C) Full-length integration of wild-type and modified PACs in transgenic mice. Shown are PCR products corresponding to rat Notch3 exon 1 (top), SP6 site (middle), and T7 site (bottom) of the PAC clone. (D) Sequence analysis of the PCR product from the rat Notch3 transgene in TgNotch3^WT (TgN3^WT) and TgNotch3^R169C (TgN3^R169C) mice. (E) RT-PCR assay of brain from nontransgenic, TgNotch3^WT, and TgNotch3^R169C (lines 88 and 92) mice. PCR products were cleaved with RsaI and fractionated on agarose gel, yielding an uncleaved 206-bp fragment from endogenous mouse Notch3 mRNA and 108- and 98-bp cleaved fragments from rat Notch3 mRNA. (F) Northern blot analysis of brain from nontransgenic, TgNotch3^WT, and TgNotch3^R169C mice. (G) Representative immunoblots of brain lysates prepared from 1-month-old TgNotch3^R169C (lines 88 and 92), TgNotch3^WT, and nontransgenic mice probed with the 5E1 anti-Notch3^ECD antibody, which recognizes endogenous mouse and exogenous rat Notch3 proteins, and the anti-SMMHC antibody. White lines indicate that the lanes were run on the same gel but were noncontiguous.