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A real-time PCR method to genotype mutant mouse models with altered affinity for cardiotonic steroids on the
Peter W Chomczynski1, Kianna M Vires1, Michal Rymaszewski1
1Molecular Research Center, Cincinnati, OH, United States of America.
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, 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 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 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.
Insights
Researchers developed a rapid PCR method to genotype mice with altered Na,K-ATPase affinity for cardiotonic steroids. This advances studies on the Na,K-ATPase receptor and its role in cell signaling and disease.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- The Na,K-ATPase enzyme, specifically its cardiotonic steroid binding site on the α subunit, is crucial for cell signaling and a therapeutic target.
- Genetically modified mouse models with altered cardiotonic steroid affinity on Na,K-ATPase α1 or α2 isoforms are valuable research tools.
- Existing genotyping methods for these models can be time-consuming.
Purpose of the Study:
- To develop a rapid and efficient genotyping method for mouse models with altered Na,K-ATPase affinity.
- To validate the new method against established techniques and ensure accuracy.
- To facilitate wider use of these valuable research models.
Main Methods:
- Development of a real-time PCR assay utilizing specific primers and fluorescent probes for mutant allele detection.
- Fast-mode PCR processing, enabling up to 15 samples in 40 minutes.
- Validation through Sanger sequencing and comparison with a previous PCR-gel electrophoresis method.
Main Results:
- A rapid, real-time PCR method for detecting mutant Na,K-ATPase alleles was successfully developed and validated.
- The new method demonstrated high efficiency, processing samples significantly faster than previous techniques.
- Sequence inconsistencies were clarified, and mutations were confirmed in the mouse colony.
Conclusions:
- The developed genotyping protocol significantly enhances the efficiency of working with Na,K-ATPase mouse models.
- Improved accessibility to these models and a streamlined genotyping process will accelerate research into Na,K-ATPase function and drug development.
- This work supports further investigation into the Na,K-ATPase cardiotonic steroid receptor's physiological roles and therapeutic potential.
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