A real-time PCR method to genotype mutant mouse models with altered affinity for cardiotonic steroids on the Na,K-ATPase

The highly conserved, cardiotonic steroid binding site (also termed ouabain binding site) on the primary alpha subunit of Na,K-ATPase plays a receptor signaling role in a range of vital cell processes and is a therapeutic target for human disease. Mouse lines with altered affinity for cardiotonic steroids on the alpha 1 or alpha 2 subunit isoform of Na,K-ATPase, without any change in pump activity, were developed by the late Jerry B Lingrel and are a valuable tool for studying its physiological roles and drug actions. In one model, the normally ouabain resistant alpha 1 isoform was rendered sensitive to ouabain binding. In a second model, the normally sensitive alpha 2 isoform was rendered resistant to ouabain binding. Additional useful models are obtained by mating these mice. To further advance their use, we developed a rapid, real-time PCR method that detects mutant alleles using specific primers and fluorescent probes. PCR is performed in fast mode with up to 15 samples processed in 40 min. The method was validated by Sanger sequencing using mice of known genotype, and by comparing results with a previous two-step method that used PCR amplification followed by gel electrophoresis. In addition, we clarified inconsistencies in published sequences, updated numbering to current reference sequences, and confirmed the continued presence of the mutations in the colony. It is expected that a wider availability of these models and a more efficient genotyping protocol will advance studies of the Na,K-ATPase and its cardiotonic steroid receptor.

Automated summary

The cardiotonic steroid binding site on Na,K-ATPase plays a vital role in cell processes and is a therapeutic target for human disease. Mouse models with altered affinity for cardiotonic steroids have been developed and a rapid genotyping method has been developed to advance their use for studying the physiological roles and drug actions.