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Generation of disease model mice by the alteration of transcription factors regulating immune responses

通过改变调节免疫反应的转录因子产生疾病模型小鼠

基本信息

批准号：
07557024
负责人：
ARAI Ken-ichi
金额：
$ 6.08万
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 (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-07557024/
关键词：
Transcription factor NF-AT STAT Immune response JAK2 JAK シグナル伝達 GM-CSF受容体 T細胞サブセット免疫応答 GM-CSF レセプター

项目摘要

The purpose of this study is to regulate the immune responses in vitro and in vivo by the alteration of the function of the transcription factors NF-AT and STAT,which are involved in the regulation of immune responses.We have isolated human NF-AT (NF-ATp/c/x) genes at the beginning of this research program, and during this study we isolated murine counterparts. Among the NF-AT family members, we focused on human and mouse NF-ATx for the analysis and alteration of its function. First, we performed domain mapping of human NF-ATx, and identified several distinct functional domains including the DNA binding domain, AP-1 interaction domain, transactivation domain CN-regulated inhibitory domain (CRI). For CRI sequence, specially deletion of this domain, resulted in nuclear translocation independent of calcium signaling. Next, we identified the calcineurin binding domain (CNBR) of mouse NF-ATx, and found that the over-expression of CNBR protein fragment inhibited NF-AT site calcineurin binding domain acted as a dominant negative mutant that prevents mNF-ATx-mediated gene activation.STAT6 has been shown to be required for mejor IL-4 responses. We constructed a conditionally active form of STAT6 by fusing STAT6 to a modified form of the hormone binding domain of the mouse estrogen receptor. This protein activates a specific receptor construct in response to the bestradiol analog 4-Hydroxy-tamoxifen. We also succeeded in the regulation of GM-CSF-induced response of BAF/3 cells by using a dominant negative mutant of JAK2, which is known to act upstream of STAT proteins.Mutant forms of NF-ATx and STAT6 derived from the above study will be useful fox the regulation of immune responses. We are now planning to generate transgenic mice expressing these mutants for the analysis of the effects of these mutants in vivo.

本研究的目的是通过改变参与免疫反应调节的转录因子 NF-AT 和 STAT 的功能来调节体外和体内的免疫反应。在本研究项目开始时，我们分离了人类 NF-AT (NF-ATp/c/x) 基因，并在本研究过程中分离了小鼠的对应基因。在 NF-AT 家族成员中，我们重点关注人类和小鼠 NF-ATx 来分析和改变其功能。首先，我们对人类 NF-ATx 进行了结构域定位，并鉴定了几个不同的功能结构域，包括 DNA 结合结构域、AP-1 相互作用结构域、反式激活结构域 CN 调节的抑制结构域 (CRI)。对于 CRI 序列，特别是删除该结构域，会导致不依赖于钙信号传导的核转位。接下来，我们鉴定了小鼠 NF-ATx 的钙调磷酸酶结合域 (CNBR)，并发现 CNBR 蛋白片段的过表达抑制了 NF-AT 位点钙调磷酸酶结合域，充当显性失活突变体，阻止 mNF-ATx 介导的基因激活。STAT6 已被证明是主要 IL-4 反应所必需的。我们通过将 STAT6 与小鼠雌激素受体的激素结合结构域的修饰形式融合，构建了 STAT6 的条件活性形式。该蛋白响应最佳二醇类似物 4-羟基-他莫昔芬，激活特定的受体结构。我们还通过使用 JAK2 的显性失活突变体成功地调节了 GM-CSF 诱导的 BAF/3 细胞反应，已知 JAK2 作用于 STAT 蛋白的上游。源自上述研究的 NF-ATx 和 STAT6 的突变形式将有助于免疫反应的调节。我们现在计划产生表达这些突变体的转基因小鼠，以分析这些突变体在体内的影响。