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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.为了建立一种新的家族性淀粉样变性多发性神经病（FAP）小鼠模型，并阐明人类变异TTR的功能，我们将人类突变体转甲状腺素（TTR）基因引入TTR缺陷小鼠。在携带人类突变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trr缺陷转基因小鼠在淀粉样蛋白沉积的发生、进展和组织分布方面有任何显著差异。这些小鼠尚未发生周围神经病变。将人类突变基因引入trr缺陷小鼠后，野生型小鼠的血清视黄醇结合蛋白（RBP）水平从3%提高到84%。野生型小鼠的血清甲状腺激素（T4）水平也从40%提高到56%。为了产生携带ttr基因点突变的小鼠，我们开发了一种新的替代型载体。该载体由ttr基因的一部分组成，在其第2外显子上携带点突变，以及一盒选择标记（MC1-neo和MC1-tk），两侧是ttr基因的第2内含子的3kb复制。将载体电穿孔到ES细胞中，从300个g418抗性克隆中分离出2个携带选择标记和ttr基因点突变的靶向克隆。从其中一个目标克隆中分离出16个fiau抗性克隆。16个无性系中有2个没有选择标记，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，获得嵌合小鼠。嵌合小鼠现在正在与C57BL/6雌性小鼠交配。这些结果表明，该载体可有效地将细微突变引入小鼠基因。