Evaluation of an FRDA-EGFP genomic reporter assay in transgenic mice

Mamm Genome. 2005 Apr;16(4):228-41. doi: 10.1007/s00335-004-3021-9.

Abstract

Friedreich ataxia is an autosomal recessive neurodegenerative disorder caused by a GAA trinucleotide expansion in the first intron of the Friedreich ataxia gene (FRDA) that causes reduced synthesis of frataxin, a mitochondrial protein likely to be involved in biosynthesis of iron-sulfur clusters. This leads to increased oxidative stress, progressive loss of large sensory neurons, and hypertrophic cardiomyopathy. To elucidate the mechanisms regulating FRDA expression and to develop an in vivo assay for agents that might upregulate FRDA expression in a therapeutically relevant manner, we have generated transgenic mice with a BAC genomic reporter construct consisting of an in-frame fusion between FRDA and the gene coding for enhanced green fluorescent protein (EGFP). Production of full-length frataxin-EGFP fusion protein was demonstrated by immunoblotting. EGFP expression was observed as early as day E3.5 of development. Most tissues of adult transgenic mice were fluorescent. The level of FRDA-EGFP expression in peripheral blood, bone marrow, and cells obtained from enzymatically disaggregated tissues was quantitated by flow cytometry. There was a twofold increase in EGFP expression in mice homozygous for the transgene when compared to hemizygous mice. These transgenic mice are a valuable tool for the examination of spatial and temporal aspects of FRDA gene expression and for the preclinical evaluation of pharmacological inducers of FRDA expression in a whole-animal model. In addition, tissues from these mice should also be valuable for stem cell transplantation studies.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromosomes, Artificial, Bacterial
  • Disease Models, Animal*
  • Evaluation Studies as Topic
  • Flow Cytometry
  • Frataxin
  • Friedreich Ataxia / genetics*
  • Gene Expression Regulation, Developmental*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • Immunoblotting
  • In Situ Hybridization, Fluorescence
  • Iron-Binding Proteins / genetics*
  • Iron-Binding Proteins / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Transgenes / genetics

Substances

  • Iron-Binding Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins