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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 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 steroi...

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Autores principales: Chomczynski, Peter W., Vires, Kianna M., Rymaszewski, Michal, Heiny, Judith A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9022855/
https://www.ncbi.nlm.nih.gov/pubmed/35446892
http://dx.doi.org/10.1371/journal.pone.0267348
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author Chomczynski, Peter W.
Vires, Kianna M.
Rymaszewski, Michal
Heiny, Judith A.
author_facet Chomczynski, Peter W.
Vires, Kianna M.
Rymaszewski, Michal
Heiny, Judith A.
author_sort Chomczynski, Peter W.
collection PubMed
description 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.
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spelling pubmed-90228552022-04-22 A real-time PCR method to genotype mutant mouse models with altered affinity for cardiotonic steroids on the Na,K-ATPase Chomczynski, Peter W. Vires, Kianna M. Rymaszewski, Michal Heiny, Judith A. PLoS One Lab Protocol 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. Public Library of Science 2022-04-21 /pmc/articles/PMC9022855/ /pubmed/35446892 http://dx.doi.org/10.1371/journal.pone.0267348 Text en © 2022 Chomczynski et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Lab Protocol
Chomczynski, Peter W.
Vires, Kianna M.
Rymaszewski, Michal
Heiny, Judith A.
A real-time PCR method to genotype mutant mouse models with altered affinity for cardiotonic steroids on the Na,K-ATPase
title A real-time PCR method to genotype mutant mouse models with altered affinity for cardiotonic steroids on the Na,K-ATPase
title_full A real-time PCR method to genotype mutant mouse models with altered affinity for cardiotonic steroids on the Na,K-ATPase
title_fullStr A real-time PCR method to genotype mutant mouse models with altered affinity for cardiotonic steroids on the Na,K-ATPase
title_full_unstemmed A real-time PCR method to genotype mutant mouse models with altered affinity for cardiotonic steroids on the Na,K-ATPase
title_short A real-time PCR method to genotype mutant mouse models with altered affinity for cardiotonic steroids on the Na,K-ATPase
title_sort real-time pcr method to genotype mutant mouse models with altered affinity for cardiotonic steroids on the na,k-atpase
topic Lab Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9022855/
https://www.ncbi.nlm.nih.gov/pubmed/35446892
http://dx.doi.org/10.1371/journal.pone.0267348
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