piggyBac-based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids
piggyBac-based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids
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DOI：
10.1111/j.1365-2583.2007.00727.x
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发表时间：
2007-06-01
影响因子：
2.6
通讯作者：
Beeman, R. W.
中科院分区：
文献类型：
--
作者：
Lorenzen, M. D.;Kimzey, T.;Beeman, R. W.
我们描述了一种有效的方法，用于产生新的piggyBac插入的生殖系的F-1杂交赤拟谷盗来自转基因辅助和供体菌株之间的杂交。辅助菌株携带编码piggyBac转座酶的单个Minos元件。供体菌株携带插入肌动蛋白基因的单个piggyBac元件，将眼睛特异性的3xP 3-EGFP（增强型绿色荧光蛋白）报告基因表达结构域扩展到包括肌肉。供体元件的再动员伴随着肌肉荧光的丧失，但眼睛荧光的保留。在中试筛选中，piggyBac供体在84%的杂交组合中被重新激活，产生了数百个新的致死、增强子陷阱、半不育和其他插入。本文所述的启动子系统使全基因组饱和插入诱变成为该鞘翅目物种中的现实目标。
We describe an efficient method for generating new piggyBac insertions in the germline of F-1 hybrid Tribolium castaneum derived from crosses between transgenic helper and donor strains. Helper strains carried single Minos elements encoding piggyBac transposase. The donor strain carried a single piggyBac element inserted into an actin gene, expanding the eye-specific, 3xP3-EGFP (enhanced green fluorescent protein) reporter expression domain to include muscle. Remobilization of the donor element is accompanied by loss of muscle fluorescence but retention of eye fluorescence. In a pilot screen, the piggyBac donor was remobilized in 84% of the hybrid crosses, generating hundreds of new lethal, enhancer-trap, semisterile and other insertions. The jumpstarter system described herein makes genome-wide, saturation insertional mutagenesis a realistic goal in this coleopteran species.