Generation of a conditional Fbw7 allele. (A) Targeting strategy showing exons 4–7 of the mouse genomic Fbw7 locus before targeting (WT allele) and after homologous recombination in C57BL/6 × 129 ES cells (targeted allele), which introduces an FRT- and LoxP-flanked PGK-Neo cassette between exons 6 and 7, and flanks exons 5 and 6 with LoxP sites. Germline removal of the PGK-Neo cassette using the FLPe-deleter strain and subsequent exposure to Cre recombinase results in excision of exons 5 and 6 and generates the conditional knockout (CKO) allele. Arrows show locations of primers (A–D) used to genotype resulting mice. (B) Before crossing to the FLPe-deleter strain, PCR using primers C and D were used to confirm transmission of the targeted allele (not depicted). After removal of the PGK-Neo cassette, primers B and C were used to detect both the “floxed” and WT alleles (497 and 315 bp, respectively). After induction of Mx-Cre with polyI-polyC injections, the floxed band was lost from the BM and thymus, and excision of exons 5 and 6 in these tissues was confirmed by PCR using primers A and C, which amplify 662 bp from the CKO allele. (C) Loss of WT Fbw7 expression was confirmed by quantitative RT-PCR using the same primers as in panel A, which are located in exons 4 and 5. Error bars show the SD of duplicate wells.