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A modified piggybac transposon system mediated by exogenous mRNA to perform gene delivery in bovine mammary epithelial cells

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Abstract

Transposons are widely used for genetic engineering in various model organisms. Recently, piggyBac (PB) has been developed as a transposable and efficient gene transfer tool in mammalian cells. In the present study, we developed three types of PB transposon systems containing a dual plasmid system (DPS), a single plasmid system (SPS), and a DNA-mRNA combined system (DRPS) and characterized their basic properties in HEK293 cells. The basic elements of the donor plasmid included a selectable-reporter gene expression cassette, two loxP sites in the same orientation, a multiple cloning site, and two chicken β-globin insulator core elements. We further identified the function of the selectable-reporter and examined PB integration sites in the human genome. Moreover, we compared the transposition efficacy and found that SPS transposed more efficiently, as compared to DPS; integration into the host genome was determined by measuring PBase activity. Results discovered the loss of PBase activity in the DRPS, indicating that this system is much more biologically safe, as compared to DPS and SPS. Finally, we employed the DRPS to successfully perform a gene delivery into bovine mammary epithelial cells (BMECs). Taken together, the information from this study will improve the flexibility of PB transposon systems and reduce the genotoxicity of PBase in genetic engineering.

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Authors and Affiliations

  1. College of Veterinary Medicine, Northwest A&F University, Yangling, 712-100, China

    Guangdong Hu, Jing Wang, Hui Huang, Fusheng Quan, Jian Kang, Yongyan Wu, Yuanpeng Gao, Feng Su, Minghao Shao & Yong Zhang

  2. Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712-100, China

    Guangdong Hu, Hui Huang, Fusheng Quan, Jian Kang, Yongyan Wu, Yuanpeng Gao, Feng Su, Minghao Shao & Yong Zhang

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  1. Guangdong Hu
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Hu, G., Wang, J., Huang, H. et al. A modified piggybac transposon system mediated by exogenous mRNA to perform gene delivery in bovine mammary epithelial cells. Biotechnol Bioproc E 19, 350–362 (2014). https://doi.org/10.1007/s12257-013-0811-5

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