In vitro Characterization of Connexin 26 Mutations

Wild-type human Cx26 (hCx26) cDNA was purchased from OriGene and was subcloned in the pGEM-HA vector (Promega) for in vitro translation. Mutations of hCx26 were produced with Quick-Change II Site-Directed Mutagenesis kits (Agilent Technologies). DNA sequencing performed at the New Jersey Medical School Molecular Resource Facility confirmed the amino acid substitutions. Nhe1-linearized hCx26 wild type, Cx43 and mutant DNAs were transcribed in vitro to cRNAs using the T7 Ultra mMessage Machine kit (Ambion). Electrophysiological data were collected using the two-electrode voltage-clamp technique as in our previous studies (Lopez et al., 2013a (link)).

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García I.E., Maripillán J., Jara O., Ceriani R., Palacios-Muñoz A., Ramachandran J., Olivero P., Pérez-Acle T., González C., Sáez J.C., Contreras J.E, & Martínez A.D. (2015). Keratitis-Ichthyosis-Deafness syndrome-associated Cx26 mutants produce nonfunctional gap junctions but hyperactive hemichannels when co-expressed with wild type Cx43. The Journal of investigative dermatology, 135(5), 1338-1347.

Publication 2015

Wild-type human Cx26 (hCx26) cDNA pGEM-HA vector Quick-Change II Site-Directed Mutagenesis kits T7 Ultra mMessage Machine kit

Amino acid substitutions Cdna Crnas Cx26 Cx43 Dnas Human Mutations Nhe1 Pgem Promega Site directed mutagenesis Vector

Corresponding Organization :

Other organizations : University of Valparaíso, Rutgers, The State University of New Jersey, Valparaiso University, Pontificia Universidad Católica de Chile

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Variable analysis

independent variables

Mutations of hCx26

dependent variables

Electrophysiological data collected using the two-electrode voltage-clamp technique

control variables

Wild-type human Cx26 (hCx26) cDNA

controls

Wild-type human Cx26 (hCx26) cDNA

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