Construction of a piggyBac

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• 作者：Keiro Uchino ; Hideki Sezutsu ; Morikazu Imamura ; Isao Kobayashi ; Ken-Ichiro Tatematsu ; Tetsuya Iizuka ; Naoyuki Yonemura ; Kazuei Mita ; Toshiki Tamura
• 关键词：Bombyx ; Enhancer trap ; Mutagenesis ; piggyBac ; Silkworm
• 刊名：Insect Biochemistry and Molecular Biology
• 出版年：2008
• 出版时间：December 2008
• 年：2008
• 卷：38
• 期：12
• 页码：1165-1173
• 全文大小：558 K

文摘

Enhancer trapping and insertional mutagenesis are powerful tools for analyzing genetic function. To construct an enhancer trap system in the silkworm Bombyx mori, we developed efficient jumpstarter strains by inserting the piggyBac transposase gene under the control of Bombyx cytoplasmic actin gene (BmA3) promoter into the genome. To stabilize the inserted transgene, the jumpstarter strains were constructed using the Minos transposon as a vector. The ability of each of the 13 jumpstarter strains to remobilize their respective transposons was tested by crossing the jumpstarters with a mutator strain carrying a GAL4 construct containing the BmA3 promoter. Four strains with high remobilization activity were then selected and used to produce enhancer trap lines by crossing with the mutator strains and hybridizing the F1 progeny with a UAS-EGFP strain. Several enhancer trap lines showing characteristic expression patterns at the embryonic, larval, pupal, and adult stages were detected in the subsequent generation. Approximately 10–40 % of the silkworms from each cross in the hybridized brood had a remobilized mutator. An analysis of the insertion positions in 105 lines by inverse PCR using a silkworm genome database revealed that remobilization occurred randomly in each chromosome. The frequency of insertion of the remobilized mutator into putative exons, introns, intergenic regions, and repetitive sequences was 12, 9, 36, and 40 % , respectively. We concluded that the piggyBac-based GAL4 enhancer trap system developed in this study is applicable for large-scale enhancer trapping in the silkworm.