Bierhuizen M F, Westerman Y, Hartong S C, Visser T P, Wognum A W, Wagemaker G
Institute of Hematology, Erasmus University Rotterdam, The Netherlands.
Leukemia. 1999 Apr;13(4):605-13. doi: 10.1038/sj.leu.2401374.
The feasibility of using the enhanced green fluorescent protein (EGFP) as a selectable reporter molecule of retroviral-mediated gene transfer in immature rhesus monkey and human CD34+ hematopoietic cells was examined. Retroviral transduction with the MFG-EGFP retroviral vector resulted in readily detectable EGFP expression in 27% of human and 11-35% of rhesus monkey bone marrow cells, and in 17-38% of rhesus monkey peripheral blood cells mobilized with FLT3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF). In addition, we used the human CD34+ KG1A cell line as a model to study viability and growth of successfully transduced cells. Cultures of mock- and EGFP-transduced KG1A cells generated equal viable cell numbers for at least 1 month, indicating the absence of a cytotoxic effect of EGFP expression in these cells. FACS selection on the basis of EGFP and CD34 expression resulted in enriched subsets (> or = 87%) of CD34+ EGFP-negative and CD34+ EGFP-positive KG1A, rhesus monkey and human bone marrow cells, demonstrating the potential of obtaining almost pure populations of transduced immature hematopoietic cells. EGFP expression was also readily demonstrated in erythroid and granulocyte/macrophage colonies derived from the CD34+ EGFP-positive rhesus monkey and human bone marrow cells by either inverted fluorescence microscopy or flow cytometry. Using four-color flow cytometry, EGFP expression could also be demonstrated in viable and phenotypically defined immature subpopulations of the CD34+ cells, ie those expressing little or no HLA-DR (rhesus monkey) or CD38 (human) antigens at the cell surface. These results demonstrate that EGFP is a very useful marker to monitor gene transfer efficiency in phenotypically defined immature rhesus monkey and human hematopoietic cell types and to select for these cells by multicolor flow cytometry prior to transplantation.
我们检测了使用增强型绿色荧光蛋白（EGFP）作为逆转录病毒介导的基因转移在未成熟恒河猴和人CD34+造血细胞中的选择报告分子的可行性。用MFG - EGFP逆转录病毒载体进行逆转录病毒转导后，27%的人骨髓细胞和11% - 35%的恒河猴骨髓细胞中可轻易检测到EGFP表达，在17% - 38%用FLT3配体（FL）和粒细胞集落刺激因子（G - CSF）动员的恒河猴外周血细胞中也可检测到。此外，我们用人CD34+ KG1A细胞系作为模型来研究成功转导细胞的活力和生长情况。未转导和EGFP转导的KG1A细胞培养物在至少1个月内产生的活细胞数量相等，表明这些细胞中EGFP表达不存在细胞毒性作用。基于EGFP和CD34表达进行荧光激活细胞分选（FACS），可富集CD34+ EGFP阴性和CD34+ EGFP阳性的KG1A、恒河猴和人骨髓细胞亚群（≥87%），证明了获得几乎纯的转导未成熟造血细胞群体的潜力。通过倒置荧光显微镜或流式细胞术也很容易在源自CD34+ EGFP阳性恒河猴和人骨髓细胞的红系和粒细胞/巨噬细胞集落中检测到EGFP表达。使用四色流式细胞术，在CD34+细胞的有活力且表型明确的未成熟亚群中，即那些细胞表面几乎不表达或不表达HLA - DR（恒河猴）或CD38（人）抗原的亚群中，也可检测到EGFP表达。这些结果表明，EGFP是一种非常有用的标志物，可用于监测表型明确的未成熟恒河猴和人造血细胞类型中的基因转移效率，并在移植前通过多色流式细胞术对这些细胞进行分选。
