Use of embryonic stem cells to introduce mutations into mice

使用胚胎干细胞将突变引入小鼠体内

基本信息

批准号：
04044111
负责人：
SHIMADA Kazunori
金额：
$ 9.41万
依托单位：
Research Institute for Microbial Diseases, Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
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

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04044111/
关键词：
ES FAP Transthyretin TTR-deficient mouse Retinol RBP Thyroid hormone Replacement type vector 血清アミロイドP成分

项目摘要

1.To generate a new mouse model of familial amyloidotic polyneuropathy (FAP) and to elucidate the function of the human variant TTR,we introduced the human mutant transthyretin (ttr) gene into the TTR-deficient mice. In the TTR-deficient mice carrying the human mutant ttr gene, amyloid began to deposit at the age 11 months. We have not yet observed any significant differences in the onset, progression and tissue distribution of amyloid deposition between the wild type and TTR-deficient transgenic mice carrying the human mutant gene. These mice have not developed peripheral neuropathy yet.2.The introduction of the human mutant gene into the TTR-deficient mice increased the serum level of retinol-binding protein (RBP) from 3% to 84% of the wild type mice. The introduction also increased the serum level of thyroid hormone (T4) from 40% to 56% of the wild type mice.3.To produce mice carrying a point mutation in the ttr gene, we developed a novel replacement type vector. This vector consists of a part of the ttr gene carrying the point mutation in its 2nd exon, and a cassette of selection markers (MC1-neo and MC1-tk) flanked with a 3 kb duplication of the 2nd intron of the ttr gene. After electroporating the vector into ES cells, 2 targeted clones carrying the selection markers as well as the point mutation in the ttr gene were isolated from 300 G418-resistant clones. Sixteen FIAU-resistant clones were isolated from one of the two targeted clones. Two of the 16 clones carried no selection markers, but retained the point mutation in the ttr gene. After injecting the targeted ES cells into C57BL/6 host blastocysts, chimeric mice were obtained. The chimeric mice were now being bred with C57BL/6 females. These results suggested that this vector is useful to introduce subtle mutations efficiently into mouse genes.

1.为了建立新的家族性淀粉样多发性神经病（FAP）小鼠模型并阐明人类变异TTR的功能，我们将人类突变转甲状腺素蛋白（ttr）基因导入TTR缺陷小鼠中。在携带人类突变型 ttr 基因的 TTR 缺陷小鼠中，淀粉样蛋白在 11 个月大时开始沉积。我们尚未观察到野生型和携带人类突变基因的 TTR 缺陷转基因小鼠之间淀粉样蛋白沉积的发生、进展和组织分布有任何显着差异。这些小鼠尚未出现周围神经病变。2.将人类突变基因引入TTR缺陷小鼠中，使视黄醇结合蛋白(RBP)的血清水平从野生型小鼠的3%增加到84%。引入还使血清甲状腺激素(T4)水平从野生型小鼠的40%增加到56%。3.为了产生携带ttr基因点突变的小鼠，我们开发了一种新型替代型载体。该载体由在其第二个外显子中携带点突变的 ttr 基因的一部分和侧翼为 ttr 基因第二个内含子的 3 kb 重复的选择标记盒（MC1-neo 和 MC1-tk）组成。将载体电转入ES细胞后，从300个G418抗性克隆中分离出2个携带选择标记以及ttr基因点突变的靶向克隆。从两个目标克隆之一中分离出 16 个 FIAU 抗性克隆。 16 个克隆中有两个不携带选择标记，但保留了 ttr 基因的点突变。将靶向ES细胞注射到C57BL/6宿主囊胚中后，获得嵌合小鼠。这些嵌合小鼠现在与 C57BL/6 雌性小鼠交配。这些结果表明该载体可用于有效地将细微突变引入小鼠基因中。