Long Dingpei, Lu Weijian, Hao Zhanzhang, Xiang Zhonghuai, Zhao Aichun
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.
Transgenic Res. 2016 Dec;25(6):795-811. doi: 10.1007/s11248-016-9970-4. Epub 2016 Jun 22.
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.
高效且可诱导的重组酶介导的DNA切除是自动删除不需要的DNA序列（包括选择标记基因）的最佳技术。然而，该方法尚未在转基因蚕中建立。为了在转基因蚕中实现高效且可诱导的FLP重组酶介导的DNA切除，构建了一种转基因靶标菌株（TTS），其包含一个侧翼为FRT的家蚕细胞质肌动蛋白3基因启动子（A3）-增强型绿色荧光蛋白（EGFP）表达盒，以及两种不同类型的FLP重组酶表达辅助菌株。然后，通过胚盘前期显微注射和有性杂交将FLP重组酶导入TTS蚕中。在TTS的后代中观察到了成功的重组酶介导的A3-EGFP表达盒的删除，FLP表达载体和FLP mRNA胚盘前期显微注射的切除效率分别为2.38%和13.3%。热休克处理后，TTS与含有热休克蛋白70（Hsp70）-FLP表达盒的辅助菌株杂交产生的切除效率在32.14%至36.67%之间，而TTS与含有A3-FLP表达盒的辅助菌株杂交产生的切除效率在97.01%至100%之间。这些结果表明，FLP/FRT系统可用于在体内实现转基因蚕中不需要的DNA序列的高效且可诱导的整合后切除。我们目前的研究将促进FLP/FRT系统在未知基因功能分析中的开发和应用，并确立转基因技术在家蚕和其他鳞翅目物种中的安全性。
