Structure Analysis of Mouse Gene Encoding Kininogens, and Construction of a Vector for Targeted Disruption of Kininogen Gene.

小鼠激肽原编码基因的结构分析以及激肽原基因靶向破坏载体的构建。

• 批准号: 10672077
• 负责人: OKAMOTO Hiroshi
• 金额: $ 2.18万
• 依托单位: Kobe Gakuin University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10672077/
• 关键词: kininogen; Kinin; Kallikrein-Kinin System; ブラジキニン; カリクレイン・キニン系; ノックアウトマウス

Mammalian expresses two types of protein precursor for kinins termed high-molecular-weight and low-molecular-weight kininogens which are coded by a single gene and produced by alternative splicing of the gene transcript. To clarify roles of the kallikrein-kinin system in the physiological and pathological conditions, steps have been taken toward the generation of mice with a "knockout" of the kininogen gene. A genomic clones of the mouse kininogen were isolated from129 strain mice using LA-PCR method and their coding sequences determed by DNA sequence analysis. A physical map of the DNA flanking this coding sequence was generated. According to the structure of kininogen gene, a vector was constructed for targeted disruption of the mouse kininogen gene in plasmid pPNT. This vector contains 7.5 Kb of DNA upstream from the bradykinin coding sequence, a neomycin resistance gene, and 2.4 Kb of DNA downstream from bradykinin coding sequence. Thus, the correct homologous recombination event will result in a chromosome in which the coding sequence for the bradykinin in mouse kininogen is replaced with the neomycin resistance gene. This targetting vector has been transfected into embryonic stem cells, and clones containing a targeted disruption of the mouse kininogen will be identified.

在哺乳动物中，激肽的前体蛋白分为高分子量激肽原和低分子量激肽原，它们由单个基因编码，通过基因转录本的选择性剪接产生。为了阐明激肽释放酶-激肽系统在生理和病理条件下的作用，已经采取步骤产生具有激肽原基因“敲除”的小鼠。用LA-PCR方法从129只品系小鼠中克隆了小鼠激肽原基因组，并对其编码序列进行了分析。生成了该编码序列侧翼的DNA的物理图谱。根据小鼠激肽原基因的结构，构建了一个质粒pPNT中小鼠激肽原基因的靶向破坏载体。该载体含有缓激肽编码序列上游的7.5Kb DNA，新霉素抗性基因，和缓激肽编码序列下游的2.4Kb DNA。因此，正确的同源重组事件将导致染色体中小鼠激肽原中缓激肽的编码序列被新霉素抗性基因取代。该靶向载体已被转染到胚胎干细胞中，并且将鉴定含有小鼠激肽原的靶向破坏的克隆。