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

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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).