TARGETED DISRUPTION OF THE MOUSE B-2 BRADYKININ RECEPTOR IN EMBRYONIC STEM-CELLS

Two mammalian genes encoding bradykinin (BK) receptors termed B-1 and B-2 have been identified by molecular cloning techniques. Some pharmacological data suggest the existence of further subtypes of the B-2 receptor. To unambiguously determine whether additional genes encoding B-2 BK receptors might exist in mammals, steps have been taken toward the generation of mice with a ''knockout'' of the BK B-2 receptor. A genomic clone of the mouse B-2 BK receptor was isolated and its coding sequence determined by DNA sequence analysis. A physical map of the DNA flanking this coding sequence was generated. A vector, pBS-KO-1, was constructed for targeted disruption of the mouse B-2 receptor gene. This vector contains 1 kb (kilobase) of DNA upstream of the mouse B-2 receptor coding sequence, a neomycin resistance gene (neo), and 5.4 kb of DNA downstream of the B-2 receptor coding sequence. Thus, the correct homologous recombination event will result in a chromosome in which the coding sequence for the mouse B-2 BK receptor is replaced with the neomycin resistance gene. pBS-KO-1 was transfected into embryonic stem cells, and clones containing a targeted disruption of the mouse B-2 BK receptor were identified.