Development of a cell model for functional and structural analysis of connexin co-expression: achieving homogeneous and inducible expression of multiple connexins in stable transfectants.
We set out to develop an in vitro cell model in which connexins 43, 40 and 45 are co-expressed in the same combinations as found in different sub-types of cardiomyocyte in vivo, using inducible promoters of the Tet-Off and Ecdysone systems. In initial studies, a heterogeneous pattern of gene expression was observed. To achieve homogeneous expression, an Internal Ribosome Entry Site (IRES) sequence was employed, ensuring that a single mRNA coded for connexin and antibiotic resistance. We then constructed plasmids that combine the inducibility of the Tet-Off and Ecdysone systems with the homogeneous expression given by the IRES constructs. These were demonstrated to give inducible and homogeneous expression. By using the reporter gene, Enhanced Green Fluorescent Protein (EGFP), it was further shown in the Tet-Off system that expression of the transfected gene was modulated homogeneously in all cells when induction was repressed. The cell model is now at a suitable stage of development for investigation of the functional correlates of the distinctive connexin co-expression found in different regions of the heart.
Halliday
Deborah
D
National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London, United Kingdom.
Dupont
Emmanuel
E
Coppen
Steven R
SR
Severs
Nicholas J
NJ
D000818:Animals; D002478:Cells, Cultured; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D004440:Ecdysone / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D013752:Tetracycline / Q000494:pharmacology; D000097003:Gap Junction alpha-5 Protein
D018031:Connexin 43; D017630:Connexins; D004440:Ecdysone; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D013752:Tetracycline; D000097003:Gap Junction alpha-5 Protein; C082920:connexin 45