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

1.我们创造了Floxed-Cx45基因座可以有条件移除的小鼠。当没有CRE时，他们是健康的。然而，心脏α-肌动蛋白-Cre介导的Cx45缺失会导致早期胚胎死亡。X-Gal染色是Cre/loxP介导的FLOXED-Cx45缺失的标志，在胚胎心肌细胞中检测到。在另一项实验中，胚胎心肌细胞在体外从Cx45缺陷的ES细胞分化而来。传导速度慢，但收缩节律快而不规则。因此，我们首次发现Cx45是胚胎早期心肌细胞所必需的缝隙连接蛋白。另一方面，平滑肌α-肌动蛋白-Cre介导的Cx45缺失产生了健康的小鼠，表明Cx45在平滑肌中不是必需的。然而，Cx45在血管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的Cx45基因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，事实似乎表明，当两个连接蛋白都丢失时，一个显著的缺陷可能会损害早期的卵子。我们正在尝试创造Cx43-KO/FLOXED-Cx45小鼠，这将使我们能够在所需的时间探索Cx43-Cx45双缺陷小鼠/细胞在所需的细胞类型中。我们已经揭开了Cx45-标记物在中枢神经系统中的详细时空表达谱。丘脑中的强表达是一个新的发现。这些数据使正在进行的合作非常令人兴奋(与哈佛医学院的David Paul教授和NIH的Cecilia Lo教授合作，创造了神经元/神经胶质/神经脊特异性的Cx45基因敲除)。

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).