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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Site-specific genomic targeting in Drosophila
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Site-specific genomic targeting in Drosophila

机译：果蝇中的特定位点基因组靶向

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摘要

Transcriptional regulation of transgenes depends upon genomic localization in higher eukaryotes. For the applied use of transgenic organisms as producers of pharmaceutically relevant proteins or as pest population control agents, a method to make transgene expression predictable is highly desirable. A targeting method that allows precise cassette replacement comprising solely genes of interest (without extraneous donor vector sequences) would be highly advantageous for insects and other multicellular organisms. In this report, we describe a method for transgene targeting to predefined chromosomal sites in Drosophila by using a transposon vector that, once integrated in the germ line, acts as an acceptor site for donor vectors. To make recombinational insertions irreversible, a FLIP recombinase-mediated cassette exchange strategy was used, and to enhance donor-target pairing, a homing sequence from the linotte locus was used. Site-specific recombinants were screened by interconvertible eye fluorescence marker phenotypes yielding, on average, targeted insertions at a frequency of 23%. The cassette exchange system provides for repetitive integrations into the same locus, allowing comparative analysis of true transgenic alleles. Furthermore, this method was used to stabilize a targeted transgene by the postintegration excision of putatively mobile transposon sequences. The genomic targeting and stabilization strategy described for Drosophila should be applicable to other insects, specifically for the goals of optimizing heterologous protein expression and enhancing ecological safety of transgenic strains intended for release in biocontrol programs.
机译：转基因的转录调控取决于高等真核生物中的基因组定位。为了将转基因生物用作药物相关蛋白的生产者或用作害虫种群控制剂，非常需要一种使转基因表达可预测的方法。允许仅包含目的基因的精确盒替换的靶向方法（无外源供体载体序列）对于昆虫和其他多细胞生物将是非常有利的。在此报告中，我们描述了一种通过使用转座子载体将转基因靶向果蝇中预定的染色体位点的方法，该转座子载体一旦整合到种系中，就成为供体载体的受体位点。为了使重组插入不可逆，使用了FLIP重组酶介导的盒交换策略，并且为了增强供体-靶点配对，使用了来自Linotte基因座的归巢序列。通过可相互转换的眼睛荧光标记物表型筛选位点特异性重组体，平均产生靶向插入的频率为23％。盒交换系统提供了重复整合到同一基因座中的功能，从而可以对真实的转基因等位基因进行比较分析。此外，该方法还用于通过整合后切除假定移动的转座子序列来稳定靶向转基因。果蝇描述的基因组靶向和稳定策略应适用于其他昆虫，特别是为了优化异源蛋白质表达和增强打算在生物防治计划中释放的转基因菌株的生态安全性。

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