权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Analysis of localization and physiological function which uses H-Ras protein myc tagging mice

H-Ras蛋白myc标记小鼠定位及生理功能分析

基本信息

批准号：
08670255
负责人：
NAKAMURA Kenji
金额：
$ 1.34万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670255/
关键词：
H-ras three ras genes ES cells gene replacement myc-tag gene targeting signal transduction mouse 胞内シグナル伝達

项目摘要

To investigate the in vivo functions and functional redundancy of the three ras genes, H-ras, N-ras and K-ras, we generated mice deficient in each of these genes. The development of H-ras homozygous mutant mice appeared to be normal. However, no one can imagine the subtle abnormality in H-ras deficient mice. Thus, it is necessary to examine the expression pattern of H-Ras proteins. In case of this kind of studies, specific antibodies against H-Ras proteins for immunohistochemical analyzes were indispensable. Thus, we intended to add a 10-amino acid epitope tag derived from human c-Myc to the C-terminal ends of H-Ras(H-Ras-myc-tag)for specific marker antigen and express them in mice by gene targeting.By two-steps of gene targeting in ES cells, we introduced H-ras-myc-tag into the mouse H-ras locus. Chimeric mice were generated by microinjection of the targeted ES cells into blastocysts. Chimeric males were mated to confirm the germline transmission of the targeted allele. Since the purpose of this project was to generate mice which express the Myc-tagged H-Ras proteins and obtain the offspring of these mice, this purpose has been successful.In situ hybridization analysis of H-, K-ras mRNA were carried out during the mouse embryonic development and mice which lack two or three ras genes simultaneously were generated. These study showed the existence of functional cooperation between the three Ras proteins in mouse development.We also intend to examine the intracellular distribution of H-Ras proteins in each organ by immunostaining the mice expreesing the Myc-tagged H-Ras proteins with an anti-Myc antibody. It is likely these analyzes will give an insight into the Ras mediated signal transduction pathway under the physiological conditions.

为了研究三种ras基因（H-ras、N-ras和K-ras）的体内功能和功能冗余，我们产生了这些基因中的每一种缺陷的小鼠。H-ras纯合突变小鼠的发育似乎是正常的。然而，没有人能想象H-ras缺陷小鼠的微妙异常。因此，有必要研究H-Ras蛋白的表达模式。在这种研究的情况下，针对H-Ras蛋白的特异性抗体用于免疫组织化学分析是必不可少的。因此，我们拟在H-Ras的C末端添加一个10个氨基酸的c-Myc表位标签（H-Ras-myc-tag），作为特异性标记抗原，并通过基因打靶的方法在小鼠体内表达。通过将靶向ES细胞显微注射到囊胚中产生嵌合小鼠。使嵌合雄性交配以确认靶向等位基因的种系传递。由于本项目的目的是产生表达Myc标记的H-Ras蛋白的小鼠并获得这些小鼠的后代，因此该目的已经成功。在小鼠胚胎发育过程中进行了H-、K-ras mRNA的原位杂交分析，并产生了同时缺失两个或三个ras基因的小鼠。这些研究表明三种Ras蛋白在小鼠发育过程中存在着功能上的协同作用，我们还打算用抗Myc抗体对表达Myc标记的H-Ras蛋白的小鼠进行免疫染色，以检测H-Ras蛋白在各器官中的胞内分布。这些分析很可能会深入了解生理条件下Ras介导的信号转导途径。