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Guangdong Hu,Jing Wang,Hui Huang,Fusheng Quan,Jian Kang,Yongyan Wu,Yuanpeng Gao,Feng Su,Minghao Shao,Yong Zhang 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.2
Transposons are widely used for geneticengineering in various model organisms. Recently, piggyBac(PB) has been developed as a transposable and efficientgene transfer tool in mammalian cells. In the present study,we developed three types of PB transposon systemscontaining a dual plasmid system (DPS), a single plasmidsystem (SPS), and a DNA-mRNA combined system (DRPS)and characterized their basic properties in HEK293 cells. The basic elements of the donor plasmid included aselectable-reporter gene expression cassette, two loxP sitesin the same orientation, a multiple cloning site, and twochicken β-globin insulator core elements. We further identifiedthe function of the selectable-reporter and examined PBintegration sites in the human genome. Moreover, wecompared the transposition efficacy and found that SPStransposed more efficiently, as compared to DPS; integrationinto the host genome was determined by measuring PBaseactivity. Results discovered the loss of PBase activity in theDRPS, indicating that this system is much more biologicallysafe, as compared to DPS and SPS. Finally, we employedthe DRPS to successfully perform a gene delivery intobovine mammary epithelial cells (BMECs). Taken together,the information from this study will improve the flexibilityof PB transposon systems and reduce the genotoxicity ofPBase in genetic engineering.
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