Expression of gap junction protein connexin43 in eukaryote cells

Objective: : To construct a vector encoding full-length c o nnexin43 cDNA and establish a high connexin-expression cell line. Metho ds: The plasmid encoding full-length connexin43 cDNA was amplified in E. coli RR1. The cDNA was reclaimed from gel and inserted into the eukaryoti c vector pED4. The recombinant transfected Chinese hamster ovary (CHO) cell usua lly expressed low levels of gap junction protein connexin43 and displayed very w eak dye coupling. Stable transfectants were selected by increasing stepwise the concentration of methotrexate (MTX) in culture medium. Results: Electrophoresis results indicated the vector was constructed. Several MTX-resis tance transfected clones were obtained indicating various amounts of connexin43 transcribed from the inserted cDNA. Immunocytochemical analysis revealed an incr ease in the amount of connexin43 immunoreactivity in the transfected cells. The level of dye coupling was also assessed and was found correlated with the amount of connexin43. Conclusion: The connexin43 cDNA inserted into the pE D4 vector can be expressed in the eukaryotic cells.