High-level transgene expression in primary human T lymphocytes and adult bone marrow CD34+ cells via electroporation-mediated gene delivery
High-level transgene expression in primary human T lymphocytes and adult bone marrow CD34+ cells via electroporation-mediated gene delivery
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DOI：
10.1038/sj.gt.3301252
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发表时间：
2000-08-01
期刊：
影响因子：
5.1
通讯作者：
Berneman, ZN
中科院分区：
文献类型：
--
作者：
Van Tendeloo, VFI;Willems, R;Berneman, ZN
设计有效的基因传递系统，在原代人血细胞中进行基因转移，对于基础造血研究和癌症基因治疗策略都具有重要意义。在这里，我们评估了电穿孔作为一种非病毒手段转染活化的人T淋巴细胞和成人骨髓（BM） CD34（+）细胞。我们描述了在预刺激的T淋巴细胞（16.3 +/- 1.3%）和e-day培养的成人BM CD34（+）细胞（29.6 +/- 4.6%）中有效传递基因的最佳培养和电穿孔参数。pha刺激的T细胞在体外培养48小时后最易接受转染，而CD3交联和白细胞介素(IL)-2刺激的T细胞在预刺激72小时后达到最大转染水平。EGFP表达的动力学分析显示，激活的T淋巴细胞在较长时间内维持高水平的转基因表达。此外，新鲜未受刺激的BM CD34（+）细胞持续转染（5.2 +/- 0.4%），细胞毒性最小(
The design of effective gene delivery systems for gene transfer in primary human blood cells is important both for fundamental hematopoiesis research and for cancer gene therapy strategies. Here, we evaluated electroporation as a nonviral means for transfection of activated human T lymphocytes and adult bone marrow (BM) CD34(+) cells. We describe optimal culture and electroporation parameters for efficient gene delivery in prestimulated T lymphocytes (16.3 +/- 1.3%), as well as e-day cultured adult BM CD34(+) cells (29.6 +/- 4.6%). PHA-stimulated T cells were most receptive for transfection after 48h of in vitro culture, while T cells stimulated by CD3 cross-linking and interleukin (IL)-2 achieved maximum transfection levels after 72 h of pre-stimulation. Kinetic analysis of EGFP expression revealed that activated T lymphocytes maintained transgene expression at high levels for a prolonged period. In addition, fresh unstimulated BM CD34(+) cells were consistently transfected (5.2 +/- 0.4%) with minimal cytotoxicity (