Generation of floxed Mule mice. (A) Targeting scheme. (1) Restriction map of the WT genomic mouse Mule locus. Probes used to verify targeting events are indicated as a, b, and c. The expected sizes of the restriction fragments are shown. B, BamHI; S, StuI. (2) Targeting vector (TV) construction. The loxP-flanked neomycin resistance gene (neo) was used as a selection marker during ES cell culture. Exons 76 and 77, which encode the HECT domain essential for Mule’s E3 ubiquitin ligase activity, were flanked by loxP sites (triangles). (3–5) The structure of the targeted Mule locus (3), the targeted locus after removal of neo (4), and the locus after deletion of the floxed exons (5) are shown, including the sizes of diagnostic restriction fragments. (B) Southern blot analysis of ES cell clones. Genomic DNA from WT E14K cells and two mutant clones (14D1 and 19C5) were digested with BamHI and hybridized with probe (a) to verify the targeting event. (C) Southern blot analysis of genomic DNA from agouti mice carrying the WT or targeting allele (Mule^3P/+) as indicated in A3. DNA was digested with StuI and hybridized with probe c to verify germline transmission. (D) Southern blot analysis of genomic DNA from mice carrying the deleted Mule allele (Mule^del/+) as indicated in A5. DNA was digested with StuI and hybridized with probe c.