A modified piggybac transposon system mediated by exogenous mRNA to perform gene delivery in bovine mammary epithelial cells
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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.
KeywordspiggyBac transposon transposase gene delivery bovine mammary epithelial cells
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- 9.Rad, R., L. Rad, W. Wang, J. Cadinanos, G. Vassiliou, S. Rice, L. S. Campos, K. Yusa, R. Banerjee, M. A. Li, J. de la Rosa, A. Strong, D. Lu, P. Ellis, N. Conte, F. T. Yang, P. Liu, and A. Bradley (2010) piggyBac transposon mutagenesis: A tool for cancer gene discovery in mice. Sci. 330: 1104–1107.CrossRefGoogle Scholar
- 11.Jang, G., S. Kim, S. Islam, W. Choi, S. Lee, W. Lee, B. Lee, J. Cho, and J. Moon (2011) Production of transgenic bovine cloned embryos using piggyBac transposition. Transg. Res. 20: 1176–1177.Google Scholar
- 18.Tsukiyama, T. (2011) Development of a simple and efficient method for generation of iPS cells by using piggybac system to screen novel culture conditions. Seikagaku The J. Japan. Biochem. Soc. 83: 855–858.Google Scholar
- 19.Tsukiyama, T., R. Asano, T. Kawaguchi, N. Kim, M. Yamada, N. Minami, Y. Ohinata, and H. Imai (2011) Simple and efficient method for generation of induced pluripotent stem cells using piggyBac transposition of doxycycline-inducible factors and an EOS reporter system. Genes to Cells: Devot. Mol. Cell. Mech. 16: 815–825.CrossRefGoogle Scholar
- 24.Urschitz, J., M. Kawasumi, J. Owens, K. Morozumi, H. Yamashiro, I. Stoytchev, J. Marh, J. A. Dee, K. Kawamoto, C. J. Coates, J. M. Kaminski, P. Pelczar, R. Yanagimachi, and S. Moisyadi (2010) Helper-independent piggyBac plasmids for gene delivery approaches: strategies for avoiding potential genotoxic effects. Proc. Nat. Acad. Sci. U. S. A. 107: 8117–8122.CrossRefGoogle Scholar
- 27.Warren, L., P. D. Manos, T. Ahfeldt, Y. H. Loh, H. Li, F. Lau, W. Ebina, P. K. Mandal, Z. D. Smith, A. Meissner, G. Q. Daley, A. S. Brack, J. J. Collins, C. Cowan, T. M. Schlaeger, and D. J. Rossi (2010) Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell. 7: 618–630.CrossRefGoogle Scholar
- 49.Chinnasamy, D., M. D. Milsom, J. Shaffer, J. Neuenfeldt, A. F. Shaaban, G. P. Margison, L. J. Fairbairn, and N. Chinnasamy (2006) Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI. Virol J. 3: 14.CrossRefGoogle Scholar
- 55.Yang, P. H., J. W. Wang, G. C. Gong, X. Z. Sun, R. Zhang, Z. Du, Y. Liu, R. Li, F. R. Ding, B. Tang, Y. P. Dai, and N. Li (2008) Cattle mammary bioreactor generated by a novel procedure of transgenic cloning for large-scale production of functional human lactoferrin. PloS one. 3.Google Scholar
- 56.Zakhartchenko, V., R. Alberio, M. Stojkovic, K. Prelle, W. Schernthaner, P. Stojkovic, H. Wenigerkind, R. Wanke, M. Duchler, R. Steinborn, M. Mueller, G. Brem, and E. Wolf (1999) Adult cloning in cattle: Potential of nuclei from a permanent cell line and from primary cultures. Mol. Reproduc. Develop. 54: 264–272.CrossRefGoogle Scholar