Enhanced Green Fluorescent Protein Is a Nearly Ideal Long-Term Expression Tracer for Hematopoietic Stem Cells, Whereas DsRed-Express Fluorescent Protein Is Not

Authors

  • Wen Tao Ph.D.,

    Corresponding author
    1. Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
    2. The Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana, USA
    3. The Walther Cancer Institute, Indianapolis, Indiana, USA
    • Walther Oncology Center, Indiana University School of Medicine, 950 West Walnut Street, Room 302, Indianapolis, Indiana 46202, USA. Telephone: 317-274-7568; Fax: 317-274-7592
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  • Barbara-Graham Evans,

    1. Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
    2. The Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana, USA
    3. The Walther Cancer Institute, Indianapolis, Indiana, USA
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  • Jing Yao,

    1. Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, USA
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  • Scott Cooper,

    1. Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
    2. The Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana, USA
    3. The Walther Cancer Institute, Indianapolis, Indiana, USA
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  • Kenneth Cornetta,

    1. Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
    2. Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, USA
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  • Christopher B. Ballas,

    1. Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, USA
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  • Giao Hangoc,

    1. Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
    2. The Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana, USA
    3. The Walther Cancer Institute, Indianapolis, Indiana, USA
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  • Hal E. Broxmeyer

    1. Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
    2. The Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana, USA
    3. The Walther Cancer Institute, Indianapolis, Indiana, USA
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Abstract

Validated gene transfer and expression tracers are essential for elucidating functions of mammalian genes. Here, we have determined the suitability and unintended side effects of enhanced green fluorescent protein (EGFP) and DsRed-Express fluorescent protein as expression tracers in long-term hematopoietic stem cells (HSCs). Retrovirally transduced mouse bone marrow cells expressing either EGFP or DsRed-Express in single or mixed dual-color cell populations were clearly discerned by flow cytometry and fluorescence microscopy. The results from in vivo competitive repopulation assays demonstrated that EGFP-expressing HSCs were maintained nearly throughout the lifespan of the transplanted mice and retained long-term multilineage repopulating potential. All mice assessed at 15 months post-transplantation were EGFP positive, and, on average, 24% total peripheral white blood cells expressed EGFP. Most EGFP-expressing recipient mice lived at least 22 months. In contrast, Discosoma sp. red fluorescent protein (DsRed)-expressing donor cells dramatically declined in transplant-recipient mice over time, particularly in the competitive setting, in which mixed EGFP- and DsRed-expressing cells were cotransplanted. Moreover, under in vitro culture condition favoring preservation of HSCs, purified EGFP-expressing cells grew robustly, whereas DsRed-expressing cells did not. Therefore, EGFP has no detectable deteriorative effects on HSCs, and is nearly an ideal long-term expression tracer for hematopoietic cells; however, DsRed-Express fluorescent protein is not suitable for these cells.

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