title: Adjusting the attB site in donor plasmid improves the efficiency of ΦC31 integrase system.
abstract: 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.
authors.affiliation: Shanghai Institute of Medical Genetics, Children's Hospital of Shanghai, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
authors.forename: Fei
authors.initials: F
authors.lastname: Xie
authors.forename: Qingwen
authors.initials: Q
authors.lastname: Ma
authors.forename: Shizhong
authors.initials: S
authors.lastname: Jiang
authors.forename: Zhaorui
authors.initials: Z
authors.lastname: Ren
authors.forename: Juan
authors.initials: J
authors.lastname: Wang
authors.forename: Shuzhen
authors.initials: S
authors.lastname: Huang
authors.forename: Fanyi
authors.initials: F
authors.lastname: Zeng
authors.forename: Yitao
authors.initials: Y
authors.lastname: Zeng
mesh_terms: D001287:Attachment Sites, Microbiological / Q000235:genetics*; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002898:Chromosomes, Human, Pair 8 / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019426:Integrases