A real-time PCR method to genotype mutant mouse models with altered affinity for cardiotonic steroids on the Na,K-ATPase v2
Year: 2022
Type: preprint
Abstract: The highly conserved, cardiotonic steroid binding site (also termed ouabain binding site) on the primary α 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 α1 or α2 subunit isoform of Na,K-ATPase, developed by the late Dr. Jerry Lingrel and collaborators, are a valuable tool for studying its physiological roles and drug actions.In one model, the normally ouabain resistant α1 isoform was rendered sensitive to ouabain binding. In a second model, the normally sensitive α2 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 amplifies digested tail clip samples without the need to isolate DNA, and 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 comparing results with a previous two-step method using PCR amplification followed by gel electrophoresis, and by Sanger sequencing. We also 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 the wider availability of these models and a more efficient genotyping protocol will advance studies of the Na,K-ATPase and its cardiotonic steroid receptor.
Institution: University of Cincinnati
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4226533721