Journal: Journal of the American Society of Nephrology : JASN
Article Title: The Connexin40 A96S Mutation Causes Renin-Dependent Hypertension
doi: 10.1681/ASN.2010101047
Figure Lengend Snippet: Generation of nonconditional Cx40A96S mice. (A) Homologous recombination of the nonconditional Cx40A96S vector with the genomic locus of the Cx40 gene leads to the recombined allele mCx40A96SNeo. Besides the 5′ and 3′ homologous regions, the vector consists of the mutated Cx40A96S sequence followed by an IRES-eGFP construct and a neomycin resistance gene flanked by frt sites. Flp-mediated deletion of the neomycin resistance gene results in the genotype mCx40A96S. (B) Southern blot analysis with an internal probe on SacI digested liver DNA from different genotypes verified the homologous recombination in Cx40A96S mice. Detection of a 5.5-kb fragment indicates the Cx40 wild-type allele, a 7-kb fragment the Cx40A96S allele, and a 8.9-kb fragment the Cx40A96SNeo allele. (C) PCR genotyping of Cx40A96S mice results in a 290-bp fragment for the Cx40 wild-type allele, a 350-bp fragment for the Cx40A96S allele, and a 680-bp fragment for the Cx40A96SNeo allele.
Article Snippet: Primary rabbit Cx40 antibodies (BioTrend, Cologne, Germany) were diluted 1:400 in blocking solution and the cells were incubated for 1 hour at room temperature.
Techniques: Homologous Recombination, Plasmid Preparation, Sequencing, Construct, Southern Blot