Transgene expression after stable transfer of a mammalian artificial chromosome into human hematopoietic cells
Transgene expression after stable transfer of a mammalian artificial chromosome into human hematopoietic cells
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DOI：
10.1016/j.exphem.2005.08.008
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发表时间：
2005-12-01
影响因子：
2.6
通讯作者：
Lasky, LC
中科院分区：
文献类型：
--
作者：
Vanderbyl, SL;Sullenbarger, B;Lasky, LC
Objective.哺乳动物人工染色体（MAC）向造血干细胞和祖细胞（HSPC）的转移为离体基因治疗提供了一种有前景的新策略，其消除了围绕病毒转导的许多问题，沿着有用于系统研究干细胞生物学和命运的独特平台。在这里，我们报告了一个卫星DNA为基础的人工染色体（ACE），在小鼠细胞，到人脐带血造血细胞的转移。利用阳离子试剂将编码人源化海肾绿色荧光蛋白（hrGFP）和博莱霉素抗性（zeo（R））的基因的GFP-Zeo-ACE转移到CD 34阳性选择的脐带血细胞中。ACE转移后，在存在或不存在博来霉素的情况下，在甲基纤维素中产生CFU-GM衍生的集落。荧光原位杂交显示，博来霉素耐药细胞表达GFP并含有完整的自主ACE。此外，当将这些平板上的细胞重新接种于甲基纤维素中时，我们观察到继发性博来霉素耐药CFU-GM衍生集落，证明CFU-GM祖细胞中稳定的染色体保留和转基因功能。据我们所知，这是第一份证明哺乳动物人工染色体转移和编码转基因在人造血细胞中稳定表达的报告。(c)2005年国际实验血液学学会。爱思唯尔公司出版
Objective. The transfer of mammalian artificial chromosomes (MACs) to hematopoietic stem and progenitor cells (HSPCs) presents a promising new strategy for ex vivo gene therapy that alleviates numerous concerns surrounding viral transduction along with a unique platform for the systematic study of stem cell biology and fate. Here we report the transfer of a satellite DNA-based artificial chromosome (an ACE), made in mouse cells, into human cord blood hematopoietic cells.Materials and Methods. A GFP-Zeo-ACE encoding the genes for humanized Renilla green fluorescence protein (hrGFP) and zeomycin resistance (zeo(R)) was transferred into CD34 positively selected cord blood cells using cationic reagents.Results. Post ACE transfer, CFU-GM-derived colonies were generated in methylcellulose in the presence or absence of bleomycin. Bleomycin-resistant cells expressed GFP and contained intact autonomous ACEs, as demonstrated by fluorescent in situ hybridization. Moreover, when the cells from these plates were replated in methylcellulose, we observed secondary bleomycin-resistant CFU-GM-derived colonies, demonstrating stable chromosome retention and transgene function in a CFU-GM progenitor.Conclusion. To our knowledge this is the first report demonstrating the transfer of a mammalian artificial chromosome and the stable expression of an encoded transgene in human hematopoietic cells. (c) 2005 International Society for Experimental Hematology. Published by Elsevier Inc.