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Yonemura, N. and Tamura, T. and Uchino, K. and Kobayashi, I. and Tatematsu, K. and Iizuka, T. and Sezutsu, H. and Muthulakshmi, M. and Nagaraju, J. and Kusakabe, T. (2012) PhiC31 integrase-mediated cassette exchange in silkworm embryos. Molecular Genetics and Genomics, 287 (9). pp. 731-739. ISSN 1617-4615

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To construct an effective site-specific integration system in the silkworm, we examined if phiC31 integrase works in silkworm embryos. As an assay system, we constructed an extrachromosomal cassette exchange reaction system between two attP sites of an acceptor plasmid and two attB sites of a donor plasmid. To evaluate the activity, integrase mRNAs synthesized from three different plasmids were used. We injected a mixture of the acceptor and donor plasmids with the mRNA synthesized in vitro from one of the three plasmids into silkworm embryos at 4–6 h after oviposition and recovered plasmid DNAs from the embryos 3 days after injection. The resultant plasmids were transformed into Escherichia coli and spread on selection medium plates containing the appropriate antibiotics. A colony-forming assay and restriction enzyme digestion of the plasmids purified from the colonies showed that the phiC31 integrase worked very efficiently in the silkworm embryos. Notably, a phiC31 integrase mRNA synthesized from two of the plasmids produced cassette exchange plasmids at a high frequency, suggesting that the mRNA can be used to construct a targeted integration system in silkworms.

Item Type: Article
Depositing User: Dr P Divakar
Date Deposited: 23 Nov 2015 09:28
Last Modified: 23 Nov 2015 09:28
URI: http://cdfd.sciencecentral.in/id/eprint/668

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