Site-specific integration with bacteriophage ΦC31 integrase.
Site-specific integration with bacteriophage ΦC31 integrase.
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与噬菌体 δC31 整合酶进行位点特异性整合。
DOI：
10.1101/pdb.prot069211
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发表时间：
2012
影响因子：
--
通讯作者：
Calos,MicheleP
中科院分区：
文献类型：
--
作者：
Hillman,RTyler;Calos,MicheleP
很少有非病毒技术存在的有效和稳定的真核基因转移和更少的仍然是广泛适用于细胞培养和整个生物体的应用。C31整合酶是一种位点特异性噬菌体重组酶，能够在多种实验和治疗条件下催化染色体转基因插入。该酶识别并催化噬菌体和细菌基因组中发现的附着基序（分别为attP和attB位点）之间的单向重组。使用ATCC31整合酶进行基因转移需要将attB序列克隆到携带转基因的质粒中。当将该修饰的质粒与整合酶表达质粒一起引入细胞中时，C31C31整合酶能够催化转基因质粒插入靶基因组中显示与野生型attP序列相似性的有限位点库中的一个。将ATCC31整合酶和attB供体质粒有效递送至感兴趣的组织或细胞仍然是该系统最具挑战性的方面。与基因组操作的病毒方法不同，使用BBC31整合酶几乎总是需要刺激细胞DNA摄取的额外方法。然而，基于质粒的系统的相对简单性意味着几乎任何经证实的将外源DNA引入细胞的方法都可以与CD31整合酶一起使用。该方案描述了在哺乳动物细胞培养物中使用CD40C31整合酶来产生显示实验转基因的稳健和稳定表达的克隆系。
Few nonviral techniques exist for efficient and stable eukaryotic gene transfer and fewer still are broadly useful in both cell culture and whole-organism applications. ɸC31 integrase, a site-specific bacteriophage recombinase, is able to catalyze chromosomal transgene insertion under a diverse range of experimental and therapeutic conditions. The enzyme recognizes and catalyzes unidirectional recombination between attachment motifs found in phage and bacterial genomes (attP and attB sites, respectively). Use of ɸC31 integrase for gene transfer requires that an attB sequence be cloned into a transgene-bearing plasmid. When this modified plasmid is introduced into cells alongside integrase-expressing plasmid, ɸC31 integrase is able to catalyze insertion of the transgene plasmid into one of a limited pool of sites in the target genome that show sequence similarity to wild-type attP. Efficient delivery of ɸC31 integrase and attB donor plasmid to the tissue or cells of interest remains the most challenging aspect of the system. Unlike viral methods of genome manipulation, use of ɸC31 integrase almost always requires an additional method of stimulating cellular DNA uptake. However, the relative simplicity of the plasmid-based system means that nearly any proven method of introducing exogenous DNA into cells can be used with ɸC31 integrase. This protocol describes the use of ɸC31 integrase in mammalian cell culture for the creation of clonal lines showing robust and stable expression of an experimental transgene.