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

为了研究H-ras、N-ras和K-ras三个ras基因在体内的功能和功能冗余，我们产生了每一个基因缺失的小鼠。H-ras纯合子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。在这类研究中，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，我们打算在H-ras的C末端增加一个来自人c-Myc的10个氨基酸的表位标签(H-ras-myc-tag)，并通过基因打靶的方法在小鼠体内表达。将靶向ES细胞显微注射到囊胚中，产生嵌合体小鼠。嵌合体雄性进行交配，以确认目标等位基因的生殖系传播。由于本项目的目的是培育表达Myc标记的H-ras蛋白的小鼠并获得这些小鼠的后代，这一目的已经成功。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，在小鼠的发育过程中，这三种Ras蛋白之间存在功能上的协同作用。我们还打算通过用抗Myc抗体对Myc标记的H-Ras蛋白进行免疫染色，来检测H-Ras蛋白在各个器官中的细胞内分布。这些分析很可能有助于深入了解在生理条件下RAS介导的信号转导途径。

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.