Figure 2.

Screening for the introduced stop codon. (A) A 676-bp fragment was amplified using revneo3b/MOPE4 primers. MOPE4 is located on the long homology arm (LA, see Fig. 1B), downstream of the deletion. Revneo3b is located inside the Neo cassette. Among the eight agouti-colored chimeras tested, three (#889, 890, and 893) were positive. The PCR product amplified by primers revneo3b and MOPE4 was sequenced to confirm the presence of the introduced stop codon and the fidelity of the amplified fragment. (B) Screening for Neo deletion. A primer set Neo deletion primer 1 (NDEL1) and NDEL2 was used to screen mice for the deletion of the Neo cassette. The PCR product for the wild-type is 233 bp. A second band with 307 bp indicates Neo deletion in the chimeras (after Neo deletion, one set of LoxP-FRT sites remain [74 bp] in the KI chimeras). (C) Screening for presence of FLP transgene. A primer set, FLP1 and FLP2, used to screen mice for presence of the FLP transgene amplified a positive product of 725 bp; (D) FLP-positive and Neo-negative mice were crossed with C57 WT to eliminate FLP transgene. KI mice #889, 890, and 893 were FLP negative with no amplification product while FLP-positive mice genomic DNA amplified 725-bp product. MW, molecular weight marker.