Construction of mouse model of human diseases by gene targeting

基因打靶构建人类疾病小鼠模型

基本信息

批准号：
04454170
负责人：
SHIMADA Kazunori
金额：
$ 4.29万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

We developed two kinds of gene targeting vectors using mouse embryonal carcinoma F9 cell and a transthyretin (ttr) gene as a model system. We used F9 cell, because it is easy to handle and has sinilar properties to embryonal stem cell, and used the ttr gene, because mutations of the human gene cause familial amyloidotic polyneuropathy.1. A vector to introduce insertional mutations : This vector consisted of a 5.9 kd ttr fragment carrying neomycin-resistance (neo) gene in the 2nd exon and a HSV-tk (herpes simplex virus thymidine kinase) gene at the 3' end. When the plasmid DNA tail at the 3' end of HSV-tk gene was longer, the efficiency of GANC selection was higher. Structues of the vector DNAs retained in non-targeted clones suggested their exonucleolytic degradation. Capping the ends of vector DNAs with hairpin-shaped oligonucleotides increased the efficiency of GANC selection.2. A vector to introduce subtle mutations : This vector consisted of the ttr fragment carrying a 3-base mutation in the 2nd exon that causes a single amino acid substitution in TTR identical to FAP patients, and a cassette of neo and HSV-tk genes flanked with a 3 kd duplication of the 2nd intron of ttr gene. In the 1st step, those clones, in which part of the endogenous ttr gene was replaced by vector DNA through homologous recombination, were selected from G418^r clones. In the 2nd step, GANC^r clones, in which the selection marker cassette had been excised by intrachromosomal recombination, were selected from the targeted G418^r clones. Interestingly, many GANC^r clones carried HSV-tk gene methylated at the promoter region. The selection marker cassette was efficiently excised in 5 random integrants of the vector DNA,indicating that this step is applicable to a wide variety of mouse genes.

我们使用小鼠胚胎癌F9细胞和转甲状腺素蛋白（ttr）基因作为模型系统开发了两种基因靶向载体。我们使用F9细胞，因为它易于处理并且具有与胚胎干细胞相似的特性，并使用ttr基因，因为人类基因的突变会导致家族性淀粉样多发性神经病。 1．引入插入突变的载体：该载体由在第二外显子中携带新霉素抗性(neo)基因的5.9kd ttr片段和在3'端携带HSV-tk(单纯疱疹病毒胸苷激酶)基因组成。当HSV-tk基因3'端的质粒DNA尾较长时，GANC选择的效率较高。保留在非靶向克隆中的载体 DNA 的结构表明它们发生了核酸外切降解。用发夹状寡核苷酸加帽载体DNA的末端提高了GANC选择的效率。2．引入细微突变的载体：该载体由在第二个外显子中携带 3 个碱基突变的 ttr 片段组成，该突变导致 TTR 中的单个氨基酸替换与 FAP 患者相同，以及两侧带有 neo 和 HSV-tk 基因的盒ttr 基因第二个内含子的 3 kd 重复。第一步，从G418^r克隆中选择那些内源ttr基因的部分通过同源重组被载体DNA取代的克隆。在第二步中，从靶向的G418 r 克隆中选择GANC r 克隆，其中选择标记盒已经通过染色体内重组被切除。有趣的是，许多 GANC^r 克隆携带在启动子区域甲基化的 HSV-tk 基因。选择标记盒在载体DNA的5个随机整合体中被有效切除，表明该步骤适用于多种小鼠基因。