Shanghai Institute of Medical Genetics, Children's Hospital of Shanghai, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
DNA Cell Biol. 2012 Jul;31(7):1335-40. doi: 10.1089/dna.2011.1590. Epub 2012 Apr 10.
ΦC31 integrase, a site-specific recombinase, can catalyze integration of circular DNA bearing attB site into pseudo attP sites in mammalian genomes. However, the integration efficiency mediated by integrase is relatively low. Our study centered on the investigation of the impact of the position, orientation, and number of attBs in the donor plasmid on the efficiency of ΦC31 integrase system. Donor plasmids bearing various types of attBs (including forward and reverse directions, tandem, and intersperse) and reporter enhanced green fluorescent protein (EGFP) were constructed. The plasmids plus helper plasmid encoding integrase were co-transfected into HeLa cells. After G418 selection, the resistant cell colonies were counted for calculating chromosomal integration frequency. EGFP expression was detected by fluorescence-activated cell sorter and enzyme-linked immunosorbent assay analysis. The results showed that efficiency of integration mediated by integrase accounted for 70% ± 7.1% of total integration events in the transfected HeLa cells. Compared with a forward orientation of attB in donor plasmid, a reverse direction of attB or interspersed attBs showed 1.5- or 2.8-fold increase in integration efficiency, respectively, while tandem attBs in donor plasmids caused a decreased efficiency of integration. We conclude that the adjustment of attB sites in donor plasmids may be of value for gene therapy and routine genetic engineering by using ΦC31 integrase system.
ΦC31 整合酶是一种位点特异性重组酶，可催化带有 attB 位点的环状 DNA 整合到哺乳动物基因组中的假 attP 位点。然而，整合酶介导的整合效率相对较低。我们的研究集中在调查供体质粒中 attB 的位置、取向和数量对 ΦC31 整合酶系统效率的影响。构建了携带各种类型 attB（包括正向和反向、串联和交错）和报告增强型绿色荧光蛋白（EGFP）的供体质粒。将带有整合酶编码辅助质粒的质粒共转染到 HeLa 细胞中。经过 G418 选择后，通过计算染色体整合频率来计数抗性细胞集落。通过荧光激活细胞分选和酶联免疫吸附分析检测 EGFP 的表达。结果表明，整合酶介导的整合效率占转染的 HeLa 细胞中总整合事件的 70%±7.1%。与供体质粒中 attB 的正向取向相比，attB 的反向取向或交错 attB 分别提高了 1.5 倍或 2.8 倍的整合效率，而供体质粒中的串联 attB 则降低了整合效率。我们得出结论，通过调整供体质粒中的 attB 位点，可能有助于利用 ΦC31 整合酶系统进行基因治疗和常规基因工程。
