title: Highly efficient and inducible DNA excision in transgenic silkworms using the FLP/FRT site-specific recombination system.
abstract: Efficient and inducible recombinase-mediated DNA excision is an optimal technology for automatically deleting unwanted DNA sequences, including selection marker genes. However, this methodology has yet to be established in transgenic silkworms. To achieve efficient and inducible FLP recombinase-mediated DNA excision in transgenic silkworms, one transgenic target strain (TTS) containing an FRT-flanked silkworm cytoplasmic actin 3 gene promoter (A3)-enhanced green fluorescent protein (EGFP) expression cassette, as well as two different types of FLP recombinase expression helper strains were generated. Then, the FLP recombinase was introduced into the TTS silkworms by pre-blastoderm microinjection and sexual hybridization. Successful recombinase-mediated deletion of the A3-EGFP expression cassette was observed in the offspring of the TTS, and the excision efficiencies of the FLP expression vector and FLP mRNA pre-blastoderm microinjection were 2.38 and 13.3 %, respectively. The excision efficiencies resulting from hybridization between the TTS and the helper strain that contained a heat shock protein 70 (Hsp70)-FLP expression cassette ranged from 32.14 to 36.67 % after heat shock treatment, while the excision efficiencies resulting from hybridization between the TTS and the helper strain containing the A3-FLP expression cassette ranged from 97.01 to 100 %. These results demonstrate that the FLP/FRT system can be used to achieve highly efficient and inducible post-integration excision of unwanted DNA sequences in transgenic silkworms in vivo. Our present study will facilitate the development and application of the FLP/FRT system for the functional analysis of unknown genes, and establish the safety of transgenic technologies in the silkworm and other lepidopteran species.
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Beibei, Chongqing, 400716, People's Republic of China.
authors.forename: Dingpei
authors.initials: D
authors.lastname: Long
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Beibei, Chongqing, 400716, People's Republic of China.
authors.forename: Weijian
authors.initials: W
authors.lastname: Lu
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Beibei, Chongqing, 400716, People's Republic of China.
authors.forename: Zhanzhang
authors.initials: Z
authors.lastname: Hao
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Beibei, Chongqing, 400716, People's Republic of China.
authors.forename: Zhonghuai
authors.initials: Z
authors.lastname: Xiang
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Beibei, Chongqing, 400716, People's Republic of China. zhaoaichun@hotmail.com.
authors.forename: Aichun
authors.initials: A
authors.lastname: Zhao
mesh_terms: D000199:Actins / Q000235:genetics*; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D001483:Base Sequence / Q000235:genetics; D012831:Bombyx; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011995:Recombination, Genetic
chemical_list: D000199:Actins; D018840:HSP70 Heat-Shock Proteins; D049452:Green Fluorescent Proteins; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase
keywords: Bombyx mori; FLP/FRT system; Safety of transgenic technologies; Site-specific excision; Transgenic silkworms