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Construction of transformed, cultured silkworm cells and transgenic silkworm using the site-specific integrase system from phage φC31

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Abstract

The Streptomyces bacteriophage, φC31, uses a site-specific integrase enzyme to perform efficient recombination. The recombination system uses specific sequences to integrate exogenous DNA from the phage into a host. The sequences are known as the attP site in the phage and the attB site in the host. The system can be used as a genetic manipulation tool. In this study it has been applied to the transformation of cultured BmN cells and the construction of transgenic Bombyx mori individuals. A plasmid, pSK-attB/Pie1-EGFP/Zeo-PASV40, containing a cassette designed to express a egfp-zeocin fusion gene, was co-transfected into cultured BmN cells with a helper plasmid, pSK-Pie1/NLS-Int/NSL. Expression of the egfp-zeocin fusion gene was driven by an ie-1 promoter, downstream of a φC31 attB site. The helper plasmid encoded the φC31 integrase enzyme, which was flanked by two nuclear localization signals. Expression of the egfp-zeocin fusion gene could be observed in transformed cells. The two plasmids were also transferred into silkworm eggs to obtain transgenic silkworms. Successful integration of the fusion gene was indicated by the detection of green fluorescence, which was emitted by the silkworms. Nucleotide sequence analysis demonstrated that the attB site had been cut, to allow recombination between the attB and endogenous pseudo attP sites in the cultured silkworm cells and silkworm individuals.

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Acknowledgments

We gratefully acknowledge the financial support of the National Basic Research Program of China (973 Program, 2012CB114600), the Specialized Research Fund for the Doctoral Program of Higher Education (20113201130002), the Key Fostering Project for Application Research of Soochow University (Q3134991) and a project funded by the Priority Academic Program of Development of Jiangsu Higher Education Institutions.

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Authors and Affiliations

  1. School of Biology & Basic Medical Science, Soochow University, Suzhou, 215123, People’s Republic of China

    Yajuan Yin, Guangli Cao, Renyu Xue & Chengliang Gong

  2. National Engineering Laboratory for Modern Silk, Soochow University, Suzhou, People’s Republic of China

    Guangli Cao, Renyu Xue & Chengliang Gong

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  1. Yajuan Yin
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  2. Guangli Cao
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  3. Renyu Xue
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  4. Chengliang Gong
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Correspondence to Chengliang Gong.

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Yajuan Yin and Guangli Cao have contributed equally to this work.

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Yin, Y., Cao, G., Xue, R. et al. Construction of transformed, cultured silkworm cells and transgenic silkworm using the site-specific integrase system from phage φC31. Mol Biol Rep 41, 6449–6456 (2014). https://doi.org/10.1007/s11033-014-3527-5

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