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 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.
Liao
Y
Y
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA.
Duling
B R
BR
D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D002478:Cells, Cultured; D002863:Chromogenic Compounds / Q000737:chemistry; D002999:Clone Cells; D018031:Connexin 43 / Q000096:biosynthesis / Q000235:genetics*; D004622:Embryo, Mammalian / Q000378:metabolism; D005697:Galactosides / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection; D019076:Transgenes; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics*
D002863:Chromogenic Compounds; D018031:Connexin 43; D005697:Galactosides; D007211:Indoles; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside