Possible cytotoxic effect of the expression of a connexin 43-LacZ fusion gene in cells of the vascular wall

Connexin 43 (Cx43) gap junctions are hypothesized to play a key role in man y 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-La c Z 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 th is dominant negative construct in cultured vascular smooth m uscle (VSM) cells and in transgenic mice. We examined the viability of cult ured VSM cells expressing the Cx43-Lac Z fusion protein under the control o f a cytomegalovirus promoter. We also selectively expressed the dominant ne gative construct in the endothelial cells of transgenic mice under the cont rol of a Tie 2 promoter. Transient transfection of cultured VSM cells led t o good initial expression of the Cx43-Lac Z fusion protein as evidenced by X-gal staining. Following 10 days of G418 selection, 300 cell clones were e xamined. None expressed the fusion protein, based on X-gal staining and Wes tern 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 f inding was the fact that expression of the Cx43-Lac Z fusion protein was no t observed in any of seven Tie 2-Cx43-Lac Z transgenic mouse lines. Moreove r, 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. Th ese data strongly suggest that the expression of the Cx43-Lac Z fusion prot ein prevents the formation of both stable clones and transgenic animals. Th is may be due to a cytotoxic effect of the dominant negative construct or t o the fact that successful cell propagation is not possible if gap junction al transmission is completely blocked. Copyright (C) 2001 S. Karger AG, Basel.