Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells
Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells
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DOI：
10.1093/nar/27.7.1674
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发表时间：
1999-04-01
影响因子：
14.9
通讯作者：
James, MR
中科院分区：
文献类型：
--
作者：
Wade-Martins, R;Frampton, J;James, MR
我们已经构建了一种可在细菌和人类细胞之间来回转移大(>；100kb)人类基因组DNA插入片段的附体穿梭载体，并可通过活细胞检测在快速分裂的人类细胞中进行追踪。该载体(P5170)是基于大肠杆菌中使用的F因子衍生的细菌人工染色体克隆载体，添加了来自EB病毒(EBV)潜在复制起点的重复序列家族。该元件在表达EBV蛋白EBNA-1的细胞中提供核保留。我们已经将一系列基因组DNA插入到p5170中，并将其导入EBNA-1(+)人细胞系。用流式细胞术定量分析外周有丝分裂的稳定性。通过不断扩大的殖民地的时间进程摄影也追踪到了伊西米亚。一个117kb的Episome保持在接近2拷贝/细胞的水平，在细胞连续生长15个月后可以不经重排地从人类细胞穿梭到细菌细胞中。此外，在没有选择的情况下，仍然可以从培养的人类细胞中拯救圣公会198天。这种可追踪的大肠杆菌/人类细胞系穿梭载体系统能够在人类细胞中携带100kb的基因组DNA，可能被证明是基因表达研究中的一个有价值的工具。
We have constructed an episomal shuttle vector which can transfer large (>100 kb) human genomic DNA inserts back and forth between bacteria and human cells and which can be tracked in rapidly dividing human cells using a live cell assay. The vector (p5170) is based on the F factor-derived bacterial artificial chromosome cloning vector used in Escherichia coli, with the addition of the family of repeats element from the Epstein-Barr virus (EBV) latent origin of replication. This element provides nuclear retention in cells expressing the EBV protein EBNA-1. We have subcloned a series of genomic DNA inserts into p5170 and transfected the constructs into an EBNA-1(+) human cell line. Episomal mitotic stability was quantitatively analysed using flow cytometry. The episomes were also tracked by time course photography of expanding colonies. A 117 kb episome was retained at similar to 2 copies/cell and could be shuttled unrearranged from the human cells into bacterial cells after 15 months of continuous cell growth. Furthermore, the episome could still be rescued from human cells cultured in the absence of selection for 198 days. Such a trackable E.coli/human cell line shuttle vector system capable of carrying >100 kb of genomic DNA in human cells could prove a valuable tool in gene expression studies.