Promiscuous Expression of H2B-GFP Transgene in Hematopoietic Stem Cells

Background: The study of adult stem cells relies on the ability to isolate them using complex combinations of markers for flow cytometry. A recent study has used a tetracycline-regulatable H2B-GFP transgenic mouse model analogous to BrdU pulse-chase methods to fluorescently label quiescent skin stem cells in vivo. In this study, we sought to use these mice to fluorescently label hematopoietic stem cells to study niche interactions. Methods and Findings: We crossed the H2B-GFP mice to mice carrying a tetracycline-regulated transactivator protein. When these mice were administered doxycycline, we observed a gradual decrease in total bone marrow GFP(+) cells over 12 weeks but the hematopoietic stem cell population remained largely GFP(+) (> 85%). In histological bone sections, the long-term GFP label-retaining cells tended to concentrate at the endosteal surface and competitive transplantation assays showed that the majority of hematopoietic stem cell activity was contained in the GFP(+) cell fraction. However, in response to stimulation with 5-fluorouracil, the hematopoietic stem cells of the crossed mice still retained a high level of GFP expression when it was anticipated the label should be lost when the cells divide. Upon further review, it was determined that the founder H2B-GFP mice showed spurious expression of the transgene at high levels in the hematopoietic stem cell population, thus the observed response of hematopoietic stem cells in the double transgenic mice to doxycycline was due to aberrant expression of the transgene and not the correct tetracycline-regulatable system. Conclusions: We observed promiscuous expression of the H2B-GFP transgene in the hematopoietic stem cell compartment of the bone marrow. This leaky expression prohibits the use of this model to study hematopoietic stem cells in vivo and careful characterization for each organ must be done if this transgenic system is to be used to isolate other prospective tissue stem cells.