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Analysis of In Vivo Mutation in the Hprt and Tk Genes of Mouse Lymphocytes

  • Vasily N. DobrovolskyEmail author
  • Joseph G. Shaddock
  • Robert H. Heflich
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 2102)

Abstract

Determining mutant frequencies in endogenous reporter genes is a tool for identifying potentially genotoxic environmental agents, and discovering phenotypes prone to genomic instability and diseases, such as cancer. Here, we describe a high-throughput method for identifying mouse spleen lymphocytes with mutations in the endogenous X-linked hypoxanthine guanine phosphoribosyl transferase (Hprt) gene and the endogenous autosomal thymidine kinase (Tk) gene. The selective clonal expansion of mutant lymphocytes is based upon the phenotypic properties of HPRT- and TK-deficient cells. The same procedure can be utilized for quantifying Hprt mutations in most strains of mice (and, with minor changes, in other mammalian species), while mutations in the Tk gene can be determined only in transgenic mice that are heterozygous for inactivation of this gene. Expanded mutant clones can be further analyzed to classify the types of mutations in the Tk gene (small intragenic mutations vs. large chromosomal mutations) and to determine the nature of intragenic mutation at both the Hprt and Tk genes.

Key words

Hypoxanthine-guanine phosphoribosyltransferase (HprtThymidine kinase (TkAlamarBlue™ Mutation Loss of heterozygosity (LOH) 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Vasily N. Dobrovolsky
    • 1
    Email author
  • Joseph G. Shaddock
    • 1
  • Robert H. Heflich
    • 1
  1. 1.Division of Genetic and Reproductive ToxicologyNational Center for Toxicological ResearchJeffersonUSA

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