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| An in vivo Robust System for Generation of Site-specific Integration Minicircle DNA Vector |
| NIE Yong-qiang1, MA Hai-yan1,2, MA Qing-wen1,2 |
1. Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200040, China;
2. Key Laboratory of Embryo Molecular Biology, Ministry of Health & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China |
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Abstract Objective: Transgenic technology has an important application value in the field of gene therapy, transgenic animals, genome modification and other biomedical researches. Many researches demonstrated that ФC31 integrase system was an efficient tool for site-specific integration, but the biggest drawback of this system is the integration of the bacterial backbone in the attB donor plasmid. The minicircle DNA is an expression vector containing only the target gene, preparation of minicircle DNA based on ФC31 integrase system is expected to avoid the introduction of bacterial backbone at the integration site, with improved biological safety of the system. The classic procedure for minicircle DNA preparation is laborious with low yield and unstable efficiency. In order to solve above problems, a feasible and effective method for obtaining site-specific integrated minicircle DNA by combining the LR clonase system and the Streptomyces phage ФC31 integrase system were established, and laying a foundation for the construction of transgenic cells without redundant sequences such as bacterial backbone. Methods: The parental plasmid containing LR clonase system and ФC31 integrase system is constructed, it can be recombined by L-arabinose induced LR clonase in bacteria to produce a miniplasmid expressing ФC31 integrase in eukaryotic cells and a minicircle DNA containing the original genes such as the target gene and attB site. The recombination efficiency was qualitatively analyzed by restriction endonuclease digestion and quantitatively analyzed by calculating the proportion of minicircle DNA, miniplasmid and the parental plasmid. The change of the ratio of minicircle DNA/miniplasmid was observed along with the induction time. Results: A validation plasmid and a parental plasmid for the production of site-specific integrated minicircle DNA were constructed.The LR clonase system could effectively catalyze the recombination of attL and attR in the parental plasmid in bacteria, and the recombination rate was more than 85%. Conclusion: A parental plasmid which can be recombined to generate site-specific integration minicircle DNA by L-arabinose induction in bacteria with higher and more stable efficiency when compare to the in vitro LR recombination were constructed, which will expand the application potential of minicircle DNA in transgenic researches.
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Received: 06 March 2017
Published: 25 July 2017
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