FIG. 2.

Removing the floxed neo cassette in vivo to generate bifloxed endoglin mice. (A) Breeding strategy showing how the neo cassette was removed from the trifloxed mice using the Meu-Cre40 transgenic line. (B) The bifloxed endoglin allele - with the neo cassette removed- was recognized by PCR for a 566 bp product with primers y and z and readily discriminated from the 411 bp product corresponding to the WT allele (upper panel). (The presence of the 1.8 kb neo cassette in the tri-floxed allele precludes the formation of a detectable PCR product.) The lower panel shows PCR products amplified with primers w and x to produce a 500 bp product from all alleles with a loxP site in intron 6. PCR products corresponding to the bifloxed allele can be seen at low levels in the mosaic (F1m) mouse, but increase to ∼50% in the F2 Eng2fl/+ mouse. (C) Diagram summarizing conversion of the trifloxed allele (Eng3fl) to the required bifloxed allele (Eng2fl), including position of diagnostic primers.