Cheng L, Du C, Murray D, Tong X, Zhang Y A, Chen B P, Hawley R G
SyStemix, Inc. Palo Alto, CA, USA.
Gene Ther. 1997 Oct;4(10):1013-22. doi: 10.1038/sj.gt.3300507.
Hematopoietic stem cells are widely recognized as attractive targets for gene therapy but current protocols to transduce these cells using recombinant retroviral vectors are inefficient. To evaluate optimization of retroviral transduction of hematopoietic stem cells and stability of gene expression in their progeny, the green fluorescent protein (GFP) was explored as a reporter. We first improved sensitivity of detection > 100-fold over that achieved previously by using a novel retroviral vector (termed MGIN) expressing a high level of an enhanced GFP gene. Primitive human hematopoietic cells bearing the CD34 surface antigen and lacking lineage differentiation markers (CD34+ Lin-) were transduced with the MGIN vector using a clinically applicable supernatant procedure. Under the conditions employed, > 75% of the target cells retained the CD34+ Lin- primitive phenotype after 4-5 days in culture, of those > or = 25% expressed a high level of GFP detectable by both flow cytometric analysis and fluorescence microscopy. When transduced cells were cultured in clonogenic progenitor assays, GFP fluorescence was readily detected in situ, indicating that GFP expression was stable and not detrimental to the differentiative potential of the transduced CD34+ Lin- cells. We conclude that GFP is effective as a vital marker to quantity retrovirus-mediated gene transfer into human hematopoietic and perhaps other types of stem/progenitor cells, and monitor gene expression during their subsequent cell lineage determinations.
造血干细胞被广泛认为是基因治疗的理想靶点，但目前使用重组逆转录病毒载体转导这些细胞的方案效率低下。为了评估造血干细胞逆转录病毒转导的优化以及其后代中基因表达的稳定性，我们探索了绿色荧光蛋白（GFP）作为报告基因。我们首先通过使用一种表达高水平增强型GFP基因的新型逆转录病毒载体（称为MGIN），将检测灵敏度提高到比之前高出100倍以上。使用临床适用的上清液程序，用MGIN载体转导携带CD34表面抗原且缺乏谱系分化标记（CD34+ Lin-）的原始人类造血细胞。在所采用的条件下，培养4-5天后，超过75%的靶细胞保留了CD34+ Lin-原始表型，其中≥25%的细胞通过流式细胞术分析和荧光显微镜检测到高水平的GFP表达。当转导细胞在克隆祖细胞测定中培养时，很容易原位检测到GFP荧光，这表明GFP表达稳定，对转导的CD34+ Lin-细胞的分化潜能没有损害。我们得出结论，GFP作为一种重要标记物，可有效定量逆转录病毒介导的基因转移到人类造血细胞以及可能的其他类型的干/祖细胞中，并在其随后的细胞谱系确定过程中监测基因表达。
