Protocol

Genetic Modification of Hematopoietic Stem Cells

Volume 506 of the series Methods In Molecular Biology™ pp 423-436

Quantification of Genomic Mutations in Murine Hematopoietic Cells

  • Hartmut GeigerAffiliated withDivision of Experimental Hematology, Cincinnati Children's Hospital Medical Center and Department of Medicine, University of Cincinnati
  • , Schleimer DavidAffiliated withDivision of Experimental Hematology, Cincinnati Children's Hospital Medical Center and Department of Medicine, University of Cincinnati
  • , Kalpana J. NattamaiAffiliated withDivision of Experimental Hematology, Cincinnati Children's Hospital Medical Center and Department of Medicine, University of Cincinnati
  • , Vijg JanAffiliated withBuck Institute for Age Research

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Summary

Maintaining the stability of the genome is critical to cell survival and normal cell growth. Genetic modification of hematopoietic cells might bear an inherent increased risk for the accumulation of DNA mutations. It frequently requires cultivation of the cells under super-physiological oxygen levels, which can result in increased oxidative damage, as well as under super-physiological concentrations of cytokines, which might interfere with DNA-damage checkpoint activation and by this means might result in an increased mutational load. We describe here a protocol for monitoring the frequency of DNA mutations in bone marrow cells post transduction or upon selection either in vitro or in vivo based on the lacZ-plasmid (pUR288) transgenic mouse (small blue mouse) mutation indicator strain.

Key words

Hematopoietic cells Gene transfer Mutation DNA damage Transgenic pUR288