Abstract
Currently, 15 different connexin genes have been described in the murine genome (1–4). Different connexins are expressed in a cell specific fashion, and in most cell types two or more connexins have been demonstrated. To characterize defined connexin channels independently of other connexins that are coexpressed in most mammalian cells, exogenous connexin DNAs can be trans-fected and expressed in cultured cells that show a low level of endogenous gap junction channels (5).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bruzzone R., White T. W., and Goodenough D. A. (1996) Connections with connexins: the molecular basis of direct intercellular signalling. Eur. J. Biochem. 238, 1–27.
Condorelli D. F., Parenti R., Spinella F., Salinaro A. T., Belluardo N., Cardile V., and Cicirata F. (1998) Cloning of a new gap junction gene (Cx36) highly expressed in mammalian brain neurons. Eur. J. Neurosci. 10, 1202–1208.
Söhl G., Degen J., Teubner B., and Willecke K. (1998) The murine gap junction gene connexin36 is highly expressed in mouse retina and regulated during brain development. FEBS Lett. 428, 27–31.
Manthey D., Bukauskas F., Lee L., Kozak C., and Willecke K. (1999) Molecular cloning and functional expression of the mouse gap junction gene connexin57 in human HeLa cells. J. Biol. Chem., 519, 631–644.
Willecke K. and Haubrich S. (1996) Connexin expression systems: To what extent do they reflect the situation in the animal? J. Bioenerg. Biomembr. 28, 319–326.
Fishman G. I., Yang G., Hertzberg E. L., and Spray D. C. (1995) Reversible intercellular coupling by regulated expression of a gap junction channel gene. CellAdhes. Commun. 3, 353–365.
Dahl G., Miller T., Paul D., Voellmy R., and Werner R. (1987) Expression of functional cell-cell channels from cloned cat liver gap junction complementary DNA. Science 236, 1290–1293.
George C. H., Martin P. E. M., and Evans W. H. (1998) Rapid determination of gap junction formation using HeLa cells microinjected with cDNAs encoding wild-type and chimeric connexins. Biochem. Biophys. Res. Commun. 247, 785–789.
Barrio L. C., Suchuyna T., Bargiello T., Xu L. X., Roginski R. S., Bennett M. L. V., and Nicholson B. J. (1991). Gap junctions formed by connexin26 and-32 clone and in combination are differently affected by applied voltage. Proc. Natl. Acad. Sci. USA 88, 8410–8414.
Holder J. W., Elmore E., and Barrett J. C. (1993) Gap junction function and cancer. Cancer Res. 53, 3475–3485.
Mesnil M., Krutovsikh V., Piccoli C., Elfgang C., Traub O., Willecke K., and Yamasaki H. (1995) Negative growth control of HeLa cells by connexin genes: connexin species specificity. Cancer Res. 55, 629–639.
Eghbali B., Kessler J. A., and Spray D. C. (1990) Expression of gap junction channels in communication-incompetent cells after stable transfection with cDNA encoding connexin32. Proc. Natl. Acad. Sci. USA 87, 1328–1331.
Zhu D., Caveney S., Kidder G. M., and Naus C. C. G. (1991) Transfection of C6 glioma cells with connexin43 cDNA: analysis of expression, intercellular coupling, and cell proliferation. Proc. Natl. Acad. Sci. USA 88, 1883–1887.
Rup D. M., Veenstra R. D., Wang H. Z., Brink P. R., and Beyer E. C. (1993) Chick connexin56, a novel lens gap junction protein. Molecular cloning and functional expression. J. Biol. Chem. 268, 706–712.
Steinberg T. H., Citivelli R., Geist S. T., Robertson A. J., Hick E., Veenstra R. D., Wang H. Z., Warlow P, M., Westphale E. M., and Liang J. G. (1994) Connexin43 and connexin45 form jap junction with different molecular permeabilities in osteoblastic cells. EMBO J. 13, 744–750.
Graham F. L. and van der Eb A. J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456–459.
McCutchan J. H. and Pegano J. S. (1968) Enhancement of the infectivity of simian virus 40 desoxyribonucleic acid with diethylaminoethyl-dextran. J. Natl. Cancer Inst. 41, 351–357.
Neumann E., Schaefer-Ridder M., Wang Y., and Hofschneider H. P. (1982) Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1, 841–845.
Fraley R. (1980) Introduction of liposome-encapsulated SV40 DNA into cells. J. Biol. Chem. 255, 10,431–10,435.
Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., and Danielsen M. (1987) Lipofection: a high efficient, lipid mediated DNA transfection procedure. Proc. Natl. Acad. Sci. USA 84, 7413–7417.
Eckert, R., Dzarlieva-Barkowskarya, A., and Hülser, D.F. (1993) Biophysical characterization of gap junction channels in HeLa cells. Pflügers Arch. 205, 404–407.
Elfgang, C., Eckert, R., Lichtenberg-Fraté, H., Butterweck, A., Traub, O., Klein, R.A., Hülser, D.F., and Willecke K. (1995) Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. J.Cell Biol. 129, 805–817.
Cao, F., Eckert, R., Elfgang, C., Nitsche, J.M., Snyder, S.A., Hülser, D.F., Willecke, K., and Nicholson, B.J. (1998) A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes. J.Cell Sci. 111, 31–43.
Haubrich, S., Schwarz, H.J., Bukauskas, F., Lichtenberg-Frate, H., Traub, O., Weingart, R., and Willecke, K. (1996) Incompatibility of connexin 40 and 43 hemichannels in gap junctions between a mammalian cells is determined by intra-cellular domains. Mol.Biol.Cell 7, 1995–2006.
Horst, M., Harth, N., and Hasilik. (1991) Biosynthesis of glycosylated human lysozyme mutants. J.Biol.Chem. 266, 13,914–13,919.
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1989–1998) Current Protocols in Molecular Biology, Vol.1–3. John Wiley & Sons, New York.
Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning:A Laboratory Manual, 2nd edit., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, NY.
Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal.Biochem. 162, 156–159.
Ishiura, M., Hirose, S., Uchida, T., Hamada, Y., Suzuki, Y., and Okada, Y. (1982) Phage particle-mediated gene transfer to cultured mammalian cells. Mol. Cell. Biol. 2, 607–616.
Chen, C.and Okayama, H. (1987) High efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7, 2745–2752.
Traub, O., Look, J., Dermietzel, R., Brümmer, F., Hülser, D., and Willecke, K. (1989) Comparative characterization of the 21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes. J.Cell Biol. 108, 1039–1051.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2001 Humana Press Inc.
About this protocol
Cite this protocol
Manthey, D., Willecke, K. (2001). Transfection and Expression of Exogenous Connexins in Mammalian Cells. In: Bruzzone, R., Giaume, C. (eds) Connexin Methods and Protocols. Methods In Molecular Biology™, vol 154. Humana Press. https://doi.org/10.1385/1-59259-043-8:187
Download citation
DOI: https://doi.org/10.1385/1-59259-043-8:187
Publisher Name: Humana Press
Print ISBN: 978-0-89603-658-1
Online ISBN: 978-1-59259-043-8
eBook Packages: Springer Protocols