Generation of EP4-deficient mice

The EP4 cDNA probe was used to isolate genomic clones from a 129/Sv mouse genomic library. Sequence analysis confirmed that these clones corresponded to the mouse EP4 gene. In the targeting vector, exons 1 and 2 are replaced by the neomycin-resistance gene from the plasmid pJNS2. These exons include the initiation codon and regions of the gene encoding six of the seven transmembrane-spanning domains and putative extracellular loops. 129/Ola derived E14Tg2a ES cells were grown, transformed and screened using standard method. A probe prepared from DNA immediately downstream of the targeted locus was used to identify targeted ES cells and later to genotype the mice generated from these ES cells. Chimaeras derived from targeted ES cells were mated with 129/SvEv or B6D2 (C57BL/6 DBA/2 F1) mice.