In vivo site-specific integration of transgene in silkworm via PhiC31 integrase-mediated cassette exchange

• Published in: Insect biochemistry and molecular biology Vol. 43; no. 11; pp. 997 - 1008
• Main Authors: Long, Dingpei, Zhao, Aichun, Xu, Longxia, Lu, Weijian, Guo, Qing, Zhang, Yang, Xiang, Zhonghuai
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.11.2013
• Subjects: Animals; Animals, Genetically Modified - genetics; Bacteriophages - enzymology; Bombyx - genetics; Bombyx mori; DNA; genetic engineering; genetic techniques and protocols; genetically modified organisms; insertional mutagenesis; Integrases - genetics; Integrases - metabolism; Mutagenesis, Insertional - methods; new methods; phiC31/att system; piggyBac; Recombination, Genetic; silkworms; Site-specific integration; site-specific recombination; Transgenes; Transgenic silkworm; transposons; Viral Proteins - genetics; Viral Proteins - metabolism; Site-specific integration; phiC31/att system; Bombyx mori; piggyBac; Transgenic silkworm
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/j.ibmb.2013.08.001

Current techniques for genetic engineering of the silkworm Bombyx mori genome utilize transposable elements, which result in positional effects and insertional mutagenesis through random insertion of exogenous DNA. New methods for introducing transgenes at specific positions are therefore needed to overcome the limitations of transposon-based strategies. Although site-specific recombination systems have proven powerful tools for genome manipulation in many organisms, their use has not yet been well established for the integration of transgenes in the silkworm. We describe a method for integrating target genes at pre-defined chromosomal sites in the silkworm via phiC31/att site-specific recombination system-mediated cassette exchange. Successful recombinase-mediated cassette exchange (RMCE) was observed in the two transgenic target strains with an estimated transformation efficiency of 3.84–7.01%. Our results suggest that RMCE events between chromosomal attP/attP target sites and incoming attB/attB sites were more frequent than those in the reciprocal direction. This is the first report of in vivo RMCE via phiC31 integrase in the silkworm, and thus represents a key step toward establishing genome manipulation technologies in silkworms and other lepidopteran species. [Display omitted] •In vivo recombinase-mediated cassette exchange (RMCE) was first achieved in Bombyx mori.•Frequencies of RMCE events are different when the incoming att sites are different.•PhiC31/att system was confirmed as a useful tool for genome manipulation in B. mori.•This is a key step toward establishing lepidoptera genome manipulation technologies.