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Molecular Anatomy of Gap Junction Expression Regulation Effect in Conditional Cx Knockout Mice

条件性 Cx 基因敲除小鼠间隙连接表达调控效应的分子解剖学

基本信息

批准号：
13470004
负责人：
SHIBATA Yosaburo
金额：
$ 9.09万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470004/
关键词：
gene targeting heart gap Junction connexin Cre loxP Cx 45 embryonic cardiac myocyte floxed : Cx mice gene trageting loxp

项目摘要

1. We have created mice whose floxed-Cx45 locus can be removed conditionally. They are healthy when Cre is absent. However, cardiac α-actin-Cre-mediated Cx45-deletion resulted in early embryonic lethality. X-gal staining, a hallmark of Cre/loxP-mediated floxed-Cx45 deletion, was detected in the embryonic cardiac myocytes. In another experiment, embryonic cardiac myocytes were differentiated from Cx45-deficient ES cells in vitro. Their conduction velocity was slow, but contraction rhythm was rapid and irregular. Therefore we, for the first time, found that Cx45 is an essential gap junction protein required for the early embryonic cardiac myocytes.2. On the other hand, smooth muscle α-actin-Cre-mediated Cx45-deletion yielded healthy mice, indicating that Cx45 is not essential in smooth muscle. However, Cx45 is so widely expressed in the smooth muscle cells that its detailed function on vascular smooth muscle remains to be clarified. We are examining how this vascular smooth muscle-specific Cx45 knockout affects the integrity and regulation of vascular tone.3. The attempt to detect Cx43-Cx45 doubly deficient mice by a micro-drop culture system was not successful. At present, the fact seemingly indicates that a significant defect may impair early eggs when both connexins are lost. We are trying to create Cx43-KO/floxed-Cx45 mice, which will enable us to explore Cx43-Cx45 doubly deficient mice/cells in desired cell types at desired timing.4. We have unraveled a detailed spatio-temporal expression profile of the Cx45-marker in the central nervous system. Strong expression in the thalamus was a novel finding. These data make ongoing collaboration very exciting (with Prof. David Paul at Harvard Medical School and Prof. Cecilia Lo at NIH, to create neuron/glia/neural crest-specific knockout of Cx45).

1.我们已经创造了小鼠，其cx45位点可以有条件地去除。当Cre不在时，它们是健康的。然而，心脏α-actin-Cre介导的Cx45缺失导致早期胚胎死亡。在胚胎心肌细胞中检测到X-gal染色，这是Cre/loxP介导的cx45缺失的标志。在另一个实验中，胚胎心肌细胞在体外从Cx45缺陷的ES细胞分化。传导速度慢，收缩节律快而不规则。因此，我们首次发现Cx45是早期胚胎心肌细胞所需的一种重要缝隙连接蛋白。另一方面，平滑肌α-actin-Cre介导的Cx45缺失产生健康小鼠，表明Cx45在平滑肌中不是必需的。然而，Cx45在平滑肌细胞中广泛表达，其在血管平滑肌中的具体功能尚不清楚。我们正在研究这种血管平滑肌特异性Cx45敲除如何影响血管张力的完整性和调节。用微滴培养系统检测Cx43-Cx45双缺陷小鼠的尝试没有成功。目前，这一事实似乎表明，当两种连接蛋白都丢失时，一个显著的缺陷可能会损害早期的卵子。我们正在尝试创建Cx43-KO/嵌合的Cx45小鼠，这将使我们能够在期望的时间在期望的细胞类型中探索Cx43-Cx45双缺陷小鼠/细胞。我们已经解开了详细的时空表达的Cx45标记在中枢神经系统。丘脑中的强表达是一个新发现。这些数据使得正在进行的合作非常令人兴奋（与哈佛医学院的大卫保罗教授和美国国立卫生研究院的塞西莉亚罗教授，创造神经元/神经胶质/神经嵴特异性敲除Cx45）。