Enhanced green fluorescent protein is a nearly ideal long-term expression tracer for hematopoietic stem cells, whereas DsRed-Express fluorescent protein is not
Enhanced green fluorescent protein is a nearly ideal long-term expression tracer for hematopoietic stem cells, whereas DsRed-Express fluorescent protein is not
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DOI：
10.1634/stemcells.2006-0553
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发表时间：
2007-03-01
期刊：
影响因子：
5.2
通讯作者：
Broxmeyer, Hal E.
中科院分区：
文献类型：
--
作者：
Tao, Wen;Evans, Barbara-Graham;Broxmeyer, Hal E.
有效的基因转移和表达示踪剂对于阐明哺乳动物基因的功能是必不可少的。在这里，我们已经确定了增强型绿色荧光蛋白（EGFP）和DsRed-Express荧光蛋白作为长期造血干细胞（HSC）表达示踪剂的适用性和意外的副作用。逆转录病毒转导的小鼠骨髓细胞表达EGFP或DsRed表达的单一或混合的双色细胞群体，清楚地辨别通过流式细胞术和荧光显微镜。体内竞争性再生试验的结果表明，表达EGFP的HSC几乎在移植小鼠的整个生命周期内保持不变，并保留了长期的多谱系再生潜力。移植后15个月评估的所有小鼠均为EGFP阳性，平均24%的外周白色血细胞表达EGFP。大多数表达EGFP的受体小鼠至少活了22个月。相反，Discosoma sp.红色荧光蛋白（DsRed）表达供体细胞在移植受体小鼠中随着时间的推移显着下降，特别是在竞争环境中，其中混合的EGFP和DsRed表达细胞共移植。此外，在有利于保存HSC的体外培养条件下，纯化的EGFP表达细胞生长健壮，而DsRed表达细胞则没有。因此，EGFP对造血干细胞没有明显的致劣作用，是一种理想的造血细胞长期表达示踪剂，而DsRed-Express荧光蛋白不适合造血干细胞。
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.