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

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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.

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Acknowledgments

We thank R. Williamson for informative discussions; D. Jamsai, M. Nefedov, and J. Vadolas for advice on DNA preparation for microinjection; and J. Scott for transgenic services. We also thank P. Farlie for assistance with microscopy and cryosectioning; P. Kalitsis and J. Perry for advice on DNA extraction from blastocysts in culture; A. Sylvain, C. Turner, J. Broughton, N. Kerr, B. McComb, and L. Kurth for animal care; and M. Bosmans and C. Pierides for technical assistance. This work was supported by grants from the Muscular Dystrophy Association (USA), the ‘Seek a Miracle’ Foundation, the Friedreich Ataxia Research Alliance (USA), and by a block grant from the National Health and Medical Research Council of Australia. We would also like to acknowledge the Friedreich’s Ataxia Associations of Victoria and New South Wales for their encouragement and support.

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Authors and Affiliations

  1. Cell and Gene Therapy Research Group, Murdoch Childrens Research Institute, Department of Paediatrics, The University of Melbourne, Royal Children’s Hospital, Flemington Road, Parkville, Victoria, 3052, Australia

    Joseph P. Sarsero, Timothy P. Holloway, Lingli Li, Samuel McLenachan, Lucille Voullaire & Panos A. Ioannou

  2. Disease Model Unit, Murdoch Childrens Research Institute, Department of Paediatrics, The University of Melbourne,Royal Children’s Hospital, Flemington Road, Parkville, Victoria, 3052, Australia

    Kerry J. Fowler & Sophie Gazeas

  3. Stem Cell Research Laboratory, Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, Victoria, 3002, Australia

    Ivan Bertoncello

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  1. Joseph P. Sarsero
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  2. Timothy P. Holloway
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  3. Lingli Li
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  9. Panos A. Ioannou
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Correspondence to Panos A. Ioannou.

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Sarsero, J.P., Holloway, T.P., Li, L. et al. Evaluation of an FRDA–EGFP genomic reporter assay in transgenic mice. Mamm Genome 16, 228–241 (2005). https://doi.org/10.1007/s00335-004-3021-9

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