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PRODUCTION OF MUTANT MICE AND ESTABLISHMENT OF EMBRYO BANK

突变小鼠的产生及胚胎库的建立

基本信息

批准号：
07558115
负责人：
YAMAMURA Kenichi
金额：
$ 12.48万
依托单位：
KUMAMOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07558115/
关键词：
ES CELL GENE TRAP RECOMBINATION EMBRYOID BODY Cre loxP 胚様体標的変異マウス

项目摘要

The efficient production of mice carrying mutations is possible by use of germline mutagenesis with chemical mutagents or X-rays. However, their application is limited, because the vast animal housing resource is required.Moreover, the positional cloning methods are required to identify these mutations. An alternative approach is mutagenesis in embryonic stem (ES) cell. Since 1989, when the first mice were derived with a locus specifically modified by homologous recombination in ES cells, these techniques have been used to mutate approximatery 1% of the total predicted number of mouse loci. However, homologous recombination is laborious and time-consuming. A more attracting approach is random insertional mutagenesis in ES cells using gene trap construct. To carry out the gene trap experiments, we developed a new screening system in which ES cells are differentiated into embryoid bodies (EB) in suspension culture. We found that the patterns of endoderm gene expression during EB development reflect the order found during mouse development in vivo. Thus, we used EB formation to search new genes. We also developed a new site-specific integration system using the Cre-lox recombination system of bacteriophage P1. The lox site is composed of an asymmetric 8 bp spacer flanked by 13 bp inverted repeats. We introduced nucleotide changes in the left 13 bp element (LE mutant lox site) or the right 13 bp element (RE mutant lox site). Recombination between the LE mutant lox site and RE mutant lox site produces a wild-type and a LE+RE mutant site that is poorly recognized by Cre, resulting in stable integration. By applying these new system to the gene trap, we isolated and determined the DNA sequences from 10 trap clones. Four of these are known genes, four are homolougous to ESTs, and two are unknown genes, but have sequence homology to ESTs. Thus, our gene trap system is quite efficient for identifying new genes and producing mutant mice.

通过使用带有化学诱变剂或X射线的种系诱变，可以有效地产生携带突变的小鼠。然而，它们的应用受到了限制，因为需要巨大的动物收容所资源，而且需要位置克隆的方法来鉴定这些突变。另一种方法是在胚胎干细胞中进行突变。自1989年以来，第一批小鼠通过ES细胞中的同源重组特异性地改变了一个基因座，这些技术已经被用来突变大约1%的小鼠预测基因座总数。然而，同源重组是费时费力的。一种更吸引人的方法是利用基因陷阱构建在ES细胞中进行随机插入突变。为了进行基因捕获实验，我们开发了一种新的筛选系统，在该系统中，ES细胞在悬浮培养中被分化为类胚体(EB)。我们发现，EB发育过程中内胚层基因的表达模式反映了小鼠在体内发育过程中的顺序。因此，我们使用EB构型来寻找新的基因。我们还利用噬菌体P1的Cre-lox重组系统开发了一种新的位点特异性整合系统。LOX位点由一个不对称的8个核苷酸的间隔区组成，两侧有13个核苷酸的反向重复序列。我们介绍了左侧13bp元件(LE突变lox位点)或右侧13bp元件(RE突变lox位点)的核苷酸变化。LE突变的lox位点和RE突变的lox位点之间的重组产生了一个野生型和一个不被Cre识别的Le+RE突变位点，从而导致稳定的整合。通过将这些新体系应用于基因捕获，我们从10个捕获克隆中分离并测定了DNA序列。其中四个是已知基因，四个与EST同源，两个是未知基因，但与EST序列同源。因此，我们的基因捕获系统在识别新基因和培育突变小鼠方面是非常有效的。