Borkowski J A, Hess J F
Department of Molecular Pharmacology and Biochemistry, Merck Research Laboratories, Rahway, NJ 07065, USA.
Can J Physiol Pharmacol. 1995 Jul;73(7):773-9. doi: 10.1139/y95-104.
Two mammalian genes encoding bradykinin (BK) receptors termed B1 and B2 have been identified by molecular cloning techniques. Some pharmacological data suggest the existence of further subtypes of the B2 receptor. To unambiguously determine whether additional genes encoding B2 BK receptors might exist in mammals, steps have been taken toward the generation of mice with a "knockout" of the BK B2 receptor. A genomic clone of the mouse B2 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 B2 receptor gene. This vector contains 1 kb (kilobase) of DNA upstream the mouse B2 receptor coding sequence, a neomycin resistance gene (neo), and 5.4 kb of DNA downstream of the B2 receptor coding sequence. Thus, the correct homologous recombination event will result in a chromosome in which the coding sequence for the mouse B2 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 B2 BK receptor were identified.
通过分子克隆技术已鉴定出两个编码缓激肽（BK）受体的哺乳动物基因，分别称为B1和B2。一些药理学数据表明B2受体还存在其他亚型。为明确确定哺乳动物中是否可能存在编码B2 BK受体的其他基因，已采取措施培育BK B2受体“敲除”小鼠。分离出小鼠B2 BK受体的基因组克隆，并通过DNA序列分析确定其编码序列。生成了该编码序列两侧DNA的物理图谱。构建了用于靶向破坏小鼠B2受体基因的载体pBS-KO-1。该载体包含小鼠B2受体编码序列上游1 kb（千碱基）的DNA、一个新霉素抗性基因（neo）以及B2受体编码序列下游5.4 kb的DNA。因此，正确的同源重组事件将导致一条染色体，其中小鼠B2 BK受体的编码序列被新霉素抗性基因取代。将pBS-KO-1转染到胚胎干细胞中，并鉴定出含有靶向破坏小鼠B2 BK受体的克隆。
