Establishment and applications of Epstein-Barr virus-based episomal vectors in human embryonic stem cells
Establishment and applications of Epstein-Barr virus-based episomal vectors in human embryonic stem cells
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DOI：
10.1634/stemcells.2005-0338
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发表时间：
2006-05-01
期刊：
影响因子：
5.2
通讯作者：
Zhang, Hongbo
中科院分区：
文献类型：
--
作者：
Ren, Caiping;Zhao, Ming;Zhang, Hongbo
人类胚胎干细胞（hES）能够无限增殖，但仍保持分化为许多细胞类型的潜力。在这里，我们报告了一个基于EB病毒（EBV）的载体系统，用于提高转染效率在hES细胞。含有oriP（EBV的潜在复制起点）的质粒可以作为附加型DNA在表达EBV核抗原1（EBNA 1）的人细胞中稳定繁殖，所述EBNA 1结合oriP并充当反式作用复制起始物。据报道，EB病毒复制子可以携带高达330千碱基对的DNA片段。使用含有增强型绿色荧光蛋白（EGFP）/嘌呤霉素抗性基因盒（沿着有或没有oriP）的质粒转染稳定表达EBNA 1的hES细胞。oriP的存在适度地增加了瞬时转染效率，更重要的是，与oriP-阴性质粒相比，它将稳定转染效率提高了约1，000倍。作为附加型DNA的oriP质粒和hES细胞中的绿色荧光蛋白表达在药物选择的存在下维持数月，并且在没有选择的情况下逐渐丧失（每细胞倍增2%-4%）。EBNA 1的存在不干扰我们测试的hES细胞特性或分化，并且可以在分化期间维持稳定的EGFP表达。除了转基因表达外，EBV载体系统还可以有效地提高hES细胞中的RNA干扰效率。因此，允许大DNA插入和持续表达转基因或小发夹RNA的EBV载体系统将增强使用hES细胞的基础和翻译研究。
Human embryonic stem (hES) cells are capable of unlimited cell proliferation yet maintain the potential to differentiate into many cell types. Here we reported an Epstein-Barr virus (EBV)-based vector system used to improve transfection efficiency in hES cells. Plasmids containing oriP, the latent replication origin of EBV, can be propagated stably as episomal DNA in human cells that express the EBV nuclear antigen 1 (EBNA1), which binds to oriP and functions as the trans-acting replication initiator. It was reported that the EBV replicon could harbor a DNA fragment of up to 330 kilobase pairs. Plasmids containing an enhanced green fluorescent protein (EGFP)/puromycin resistance gene cassette along with or without oriP were used to transfect hES cells that stably express EBNA1. The presence of oriP moderately increased the transient transfection efficiency and more importantly it elevated the stable transfection efficiency by approximately 1,000-fold as compared with oriP-minus plasmids. The oriP plasmid as episomal DNA and green fluorescent protein expression in hES cells was maintained for months in the presence of drug selection and gradually lost (2%-4% per cell doubling) in the absence of selection. The presence of EBNA1 did not interfere with the hES cell properties or differentiation we tested and could maintain stable EGFP expression during differentiation. In addition to transgene expression, the EBV vector system could effectively enhance the RNA interference efficiency in hES, cells. Thus, the EBV vector system that allows a large DNA insert and sustained expression of transgene or small hairpin RNA will enhance basic and translational research using hES cells.