EGFP Is a Nearly Ideal Long-term Expression Tracer for Hematopoietic Stem Cells while DsRed-Express Fluorescent Protein Is Not

¹ Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana; The Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana; he Walther Cancer Institute, Indianapolis, Indiana
² Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana
³ Microbiology and Immunology, Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana

^* To whom correspondence should be addressed. E-mail: wetao{at}iupui.edu.

	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 EGFP and DsRed-Express fluorescent protein as expression tracers in long-term hematopoietic stem cells (HSC). Retroviral 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 HSC 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, DsRed-expressing donor cells dramatically declined in transplant recipient mice over time, particularly in the competitive setting where mixed EGFP- and DsRed-expressing cells were co-transplanted. Moreover, under in vitro culture condition favoring preservation of HSC, purified EGFP-expressing cells grew robustly while DsRed-expressing cells did not. Therefore, EGFP has no detectable deteriorative effects on HSC, 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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