Groth A C, Olivares E C, Thyagarajan B, Calos M P
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.
Proc Natl Acad Sci U S A. 2000 May 23;97(11):5995-6000. doi: 10.1073/pnas.090527097.
The integrase from the Streptomyces phage phiC31 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 phiC31 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.
来自链霉菌噬菌体phiC31的整合酶能在噬菌体基因组中的attP位点与宿主细菌染色体中的attB位点之间高效重组。在本文中，我们证明该酶在人类细胞中也能发挥作用。构建了一个质粒检测系统，用于检测attP到attB的分子内整合。该检测方法用于证明，在phiC31整合酶存在的情况下，精确的单向整合在大肠杆菌中效率为100%，在人类细胞中效率大于50%。该检测系统还用于确定attB和attP的最小尺寸，分别为34 bp和39 bp。此外，在人类细胞中记录了携带attP的导入质粒与携带attB的已建立的爱泼斯坦-巴尔病毒质粒之间精确而高效的分子间整合。这项工作证明了在哺乳动物细胞中高效、位点特异性、单向整合的存在。这些观察结果为位点特异性整合策略奠定了基础，这些策略可能在广泛的基因工程应用中有用。
