Liao Y, Duling B R
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA.
J Vasc Res. 2001 May-Jun;38(3):203-11. doi: 10.1159/000051048.
Connexin 43 (Cx43) gap junctions are hypothesized to play a key role in many aspects of vascular function. In an effort to evaluate the importance of connexins in vascular function we took advantage of the fact that a Cx43-LacZ fusion protein has been reported to effectively reduce dye transfer in NIH 3T3 fibroblasts by acting as a dominant negative construct. We explored the use of this dominant negative construct in cultured vascular smooth muscle (VSM) cells and in transgenic mice. We examined the viability of cultured VSM cells expressing the Cx43-LacZ fusion protein under the control of a cytomegalovirus promoter. We also selectively expressed the dominant negative construct in the endothelial cells of transgenic mice under the control of a Tie 2 promoter. Transient transfection of cultured VSM cells led to good initial expression of the Cx43-LacZ fusion protein as evidenced by X-gal staining. Following 10 days of G418 selection, 300 cell clones were examined. None expressed the fusion protein, based on X-gal staining and Western blot analysis, but all contained the transgene, based on PCR analysis. The fusion protein was expressed in a few isolated cells, suggesting that cell division was inhibited by the fusion protein. In agreement with this finding was the fact that expression of the Cx43-LacZ fusion protein was not observed in any of seven Tie 2-Cx43-LacZ transgenic mouse lines. Moreover, a very low yield of mice carrying the transgene was observed (7/136; 5.1%). Analysis of 65 embryos at embryonic day 11.5 showed similar results. These data strongly suggest that the expression of the Cx43-LacZ fusion protein prevents the formation of both stable clones and transgenic animals. This may be due to a cytotoxic effect of the dominant negative construct or to the fact that successful cell propagation is not possible if gap junctional transmission is completely blocked.
连接蛋白43（Cx43）间隙连接被认为在血管功能的许多方面发挥关键作用。为了评估连接蛋白在血管功能中的重要性，我们利用了这样一个事实：据报道，Cx43-LacZ融合蛋白作为一种显性负性构建体，可有效减少NIH 3T3成纤维细胞中的染料转移。我们探索了这种显性负性构建体在培养的血管平滑肌（VSM）细胞和转基因小鼠中的应用。我们检测了在巨细胞病毒启动子控制下表达Cx43-LacZ融合蛋白的培养VSM细胞的活力。我们还在Tie 2启动子控制下，在转基因小鼠的内皮细胞中选择性表达显性负性构建体。培养的VSM细胞的瞬时转染导致Cx43-LacZ融合蛋白的良好初始表达，X-gal染色证明了这一点。经过10天的G418筛选后，检查了300个细胞克隆。根据X-gal染色和蛋白质印迹分析，没有一个克隆表达融合蛋白，但根据PCR分析，所有克隆都含有转基因。融合蛋白在一些孤立的细胞中表达，这表明细胞分裂受到融合蛋白的抑制。与这一发现一致的是，在七个Tie 2-Cx43-LacZ转基因小鼠品系中，没有一个观察到Cx43-LacZ融合蛋白的表达。此外，观察到携带转基因的小鼠产量非常低（7/136；5.1%）。对胚胎第11.5天的65个胚胎进行分析，结果相似。这些数据强烈表明，Cx43-LacZ融合蛋白的表达阻止了稳定克隆和转基因动物的形成。这可能是由于显性负性构建体的细胞毒性作用，或者是由于如果间隙连接传递被完全阻断，成功的细胞增殖是不可能的。
