Bierhuizen M F, Westerman Y, Visser T P, Dimjati W, Wognum A W, Wagemaker G
Institute of Hematology, Erasmus University Rotterdam, Rotterdam, The Netherlands.
Blood. 1997 Nov 1;90(9):3304-15.
The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.
能够快速检测并有效筛选成功转导细胞的标记物，将极大地促进向造血干细胞及其直接子代细胞进行基因转移的进一步改进。为此目的，设计并测试了一种逆转录病毒载体，其编码重组的维多利亚多管水母绿色荧光蛋白，该蛋白在哺乳动物细胞中经增强可实现高水平表达（增强型绿色荧光蛋白，EGFP）。用这种逆转录病毒载体转导的小鼠细胞系（NIH 3T3、Rat2）和骨髓细胞表现出稳定的绿色荧光信号，通过流式细胞术易于检测。对逆转录病毒转导的骨髓细胞进行的功能分析表明，增强型绿色荧光蛋白在体外克隆形成祖细胞（粒-巨噬细胞集落形成单位，GM-CFU）、第13天的脾集落形成单位（CFU-S）以及移植小鼠的外周血细胞和骨髓重建细胞中均有表达。结合荧光激活细胞分选（FACS）技术，增强型绿色荧光蛋白表达可作为一种标记物，用于筛选纯度超过95%的转导细胞群体，并使用针对特定细胞表面抗原的抗体从表型上定义转导细胞。未观察到增强型绿色荧光蛋白表达的有害影响：荧光强度似乎稳定，造血细胞生长未受损害。数据表明，使用增强型绿色荧光蛋白作为一种方便快捷的报告基因来监测逆转录病毒介导的基因在造血细胞中的转移和表达、筛选基因修饰细胞以及在体外和体内追踪这些细胞及其子代具有可行性。
