Abstract

In the post-genome era, precise functions of the vast majority of human and mouse genes and their interactions remain to be elucidated and defined. To assess the contributions of many different genes to hematopoiesis and to determine how they function in hematopoietic stem and progenitor cells, often requires introduction of the gene of interests or its derivative mutants into these cells along with a marker gene which is used to track the transduced cells over time. An ideal expression tracer should be easy to track and non-toxic to cells with minimal perturbation of cell metabolism. Although the enhanced green fluorescent protein (EGFP) has been widely used as an expression tracer and other fluorescent proteins were occasionally used in this capacity as well, their suitability for long-term marking of hematopoietic stem cells and their unintended side-effects on the functions of these cells have not been systematically investigated. In this study, we have performed a series of in vitro and in vivo experiments to evaluate whether two fluorescent proteins, EGFP and DsRed-Express which is an optimized variant of a red fluorescent protein from coral, are suitable for use as expression tracers in hematopoietic stem and progenitor cells. We first constructed a pair of MSCV based retroviral vectors with an identical backbone expressing either EGFP or DsRed-Express. These vectors were intended to be used for multicolor tracking of separate genes simultaneously and accurately in a single cell or mouse since the emission spectra of EGFP and DsRed-Express have minimal overlap. We used these vectors to transduce mouse mononuclear bone marrow cells, and the results demonstrated that the EGFP vector transduced green cells and DsRed-Express vector transduced red cells from single color or mixed dual color cell populations are clearly discerned by flow cytometry and fluorescent microscopy. Our results from in vivo competitive repopulation assay showed that under the experimental condition, mouse hematopoietic stem cells expressing EGFP alone are maintained nearly throughout the lifespan of the transplanted mice and appear to function normally. About 15 months after bone marrow transplantation, on average, 24% total peripheral white blood cells in recipient mice expressed EGFP. This initial donor population prior to injection contained 25.2% EGFP positive cells and all 5 mice assessed at 15 months were EGFP positive. Most EGFP transplanted mice lived at least 22 months and appeared normal at sacrifice. In contrast, the percentage of DsRed expressing donor cells transplanted either alone or mixed with EGFP expressing cells unexpectedly declined in recipient mice over time. By 3 months post-transplantation, the decrease of the percentage of DsRed expressing cells was dramatic. Therefore, EGFP itself has no detectable deteriorative effects on hematopoietic stem cells and is nearly an ideal long-term expression tracer for hematopoietic cells. However, the number of detectable DsRed expressing hematopoietic stem and progenitor cells, for reasons not yet known, decreases over time; therefore, DsRed fluorescent protein should not be used as a long-term tracer for these cells. This study also points out the importance of using correct expression tracers for accurately defining the functions of any genes.

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