Development of new technology for transgene methods in mice

小鼠转基因方法新技术的开发

基本信息

批准号：
06558114
负责人：
NABESHIMA Yo-ichi
金额：
$ 9.92万
依托单位：
National Institute of Neuroscience, NCNP
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06558114/
关键词：
Homologous Recombination ES cell Cre-recombinase loxP sequence Premature aging Insertion mutation 発生工学相同組み換 Creリコンビネース lox P 標的遺伝子組み換 loxP

项目摘要

Application of Cre-loxP system for gene engineering in mice.To develop the technology required for tissue and stage restricted gene disruption in mice, we tried the application of Cre-loxP system and obtained following results. At first, we constructed the gene targeting vector which had three loxP sequences at just 5' and 3' ends of Neo-TK cassette (inserted at upstream of the target sequence) and at downstream of the target sequence. Then, homologous recombinant ES cells were isolated and the Neo-TK cassette was removed off by the expression of Cre-recombinase. These recombinant ES cells were injected into fertilized eggs to generate chimeric mice. The histological and functional analyzes of hetero and homozygous mutant mice are now in progress.2) Isolation and analysis of premature aging mice.We recently developed a unique short life mouse strain in which a single gene mutation caused a premature aging, including arteriosclerosis, osteoporosis, impaired spermatogenesis and oocyte maturation, soft tissue calcification, atrophy of thymus, pulmonary emphysema, degeneration of Purkinje cells, and impaired glucose metabolism.However, the mice did not show any abnormality in lipid metabolisms. We have isolated the causative gene from both mouse and human. Furthermore, the ectopic expression of the isolated gene successively rescued the mutant phenotypes. The gene has been found to be a novel one encoding a membrane protein and expressed mainly in kidney and brain. In human a secreted protein, in adition to membrane protein, is generated from same gene by alternative splicing mechanism.

Cre-loxP系统在小鼠基因工程中的应用为了开发小鼠组织和阶段限制性基因破坏所需的技术，我们尝试了Cre-loxP系统的应用，并获得了以下结果。首先，我们构建了基因打靶载体，该载体在Neo-TK盒的5'和3'端（插入靶序列的上游）和靶序列的下游具有三个loxP序列。然后，分离同源重组ES细胞，并通过Cre重组酶的表达去除Neo-TK盒。这些重组ES细胞被注射到受精卵中以产生嵌合小鼠。杂合子和纯合子突变小鼠的组织学和功能分析正在进行中。2）早衰小鼠的分离和分析。我们最近开发了一种独特的短命小鼠品系，其中单基因突变导致过早衰老，包括动脉硬化、骨质疏松症、精子发生和卵母细胞成熟受损、软组织钙化、胸腺萎缩、肺气肿、浦肯野细胞变性以及糖代谢受损。然而，小鼠并没有表现出任何症状。脂质代谢异常。我们已经从小鼠和人类身上分离出了致病基因。此外，分离基因的异位表达成功挽救了突变表型。该基因被发现是一种编码膜蛋白的新基因，主要在肾脏和大脑中表达。在人类中，除了膜蛋白之外，分泌蛋白也是通过选择性剪接机制从同一基因产生的。