Generation of Ep2–/– mice

Genomic clones were isolated from a 129/Sv mouse genomic library with an Ep2 cDNA probe and their identity confirmed by sequence analysis. A targeting vector was constructed in which the DNA encoding amino acids 246–601 was replaced by a neomycin-resistant gene and electroporated into 129/Ola–derived E14TG2a embryonic stem (ES) cells, and neomycin- and ganciclovir-resistant colonies were identified using standard methods. DNA isolated from ES cell colonies was digested with the restriction enzyme HindIII and analyzed by Southern blot to identify clones with a targeted Ep2 allele. Chimeras derived from targeted ES cells were mated with 129/SvEv mice, and offspring carrying the targeted allele were identified by Southern blot analysis. These heterozygotes were intercrossed to produce mice homozygous for the Ep2 mutation.