A phage integrase directs efficient site-specific integration in human cells
A phage integrase directs efficient site-specific integration in human cells
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DOI：
10.1073/pnas.090527097
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发表时间：
2000-05-23
影响因子：
11.1
通讯作者：
Calos, MP
中科院分区：
文献类型：
--
作者：
Groth, AC;Olivares, EC;Calos, MP
来自链霉菌噬菌体phi C31的整合酶在噬菌体基因组中的attP位点和宿主细菌染色体中的attB位点之间进行有效的重组。构建了测量attP到attB中的分子内整合的质粒测定系统。该测定用于证明在phi C31整合酶的存在下，精确的单向整合在大肠杆菌中以100%的效率发生，在人细胞中以>50%的效率发生。该测定系统还用于分别在34bp和39bp处限定attB和attP的最小大小。此外，在人细胞中记录了携带attP的输入质粒精确且有效地分子间整合到已建立的携带attB的EB病毒质粒中。这项工作证明了哺乳动物细胞中高效、位点特异性、单向整合。这些观察结果形成了在广泛的基因工程应用中潜在有用的位点特异性整合策略的基础。
The integrase from the Streptomyces phage phi C31 carries out efficient recombination between the attP site in the phage genome and the attB site in the host bacterial chromosome, In this paper, we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular integration of attP into attB, This assay was used to demonstrate that in the presence of the phi C31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells. This assay system was also used to define the minimal sizes of attB and attP at 34 bp and 39 bp, respectively. Furthermore, precise and efficient intermolecular integration of an incoming plasmid bearing attP into an established Epstein-Barr virus plasmid bearing attB was documented in human cells. This work is a demonstration of efficient, site-specific, unidirectional integration in mammalian cells. These observations form the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.