Figure 3.
Generation of EP2lox/+ mice. A, Recombineering strategy to introduce loxP sites 5′ and 3′ to exon 1 of Ptger2 is diagrammed (see Materials and Methods). B, Diagrams are shown of the targeting vector with EcoRI (R) restriction sites before and after addition of recombinant Cre recombinase. Restriction map analysis of EcoRI digested targeting vector shows correct restriction digest after addition of Cre recombinase, and confirms functionality of loxP sites 5′ and 3′ to exon 1. C, PCR strategy for the identification of positive ES clones and validation of the correct insertion of the target sequences into the Ptger2 locus are shown. The 5′ loxP PCR fragment (in purple; 700 bp for floxed EP2 and 542 bp for wt) spans the inserted loxP site 5′ of Exon 1. The Exon-Neo junction (in green; 900 bp) spans the 3′ end of Exon 1 to the 5′ end of Neo. The 5′ junction PCR product (in red; 4.5 kb) spans flanking 5′ genomic sequences, the 5′ arm of the targeting construct, and the 5′ loxP site. The 3′ junction (in blue; 3.9 kb) spans the 3′ end of Neo through the 3′ arm of the targeting construct and the 3′ flanking genomic sequence. All PCR fragments were completely sequenced to confirm correct recombination and integration.