PhiC31 integrase-mediated cassette exchange in silkworm embryos

N. Yonemura, T. Tamura, K. Uchino, I. Kobayashi, K. Tatematsu, T. Iizuka, H. Sezutsu, M. Muthulakshmi, J. Nagaraju, T. Kusakabe

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)731-739
Number of pages9
JournalMolecular Genetics and Genomics
Volume287
Issue number9
DOIs
Publication statusPublished - Sep 1 2012

    Fingerprint

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

Cite this

Yonemura, N., Tamura, T., Uchino, K., Kobayashi, I., Tatematsu, K., Iizuka, T., ... Kusakabe, T. (2012). PhiC31 integrase-mediated cassette exchange in silkworm embryos. Molecular Genetics and Genomics, 287(9), 731-739. https://doi.org/10.1007/s00438-012-0711-y