Dietzl Georg, Chen Doris, Schnorrer Frank, Su Kuan-Chung, Barinova Yulia, Fellner Michaela, Gasser Beate, Kinsey Kaolin, Oppel Silvia, Scheiblauer Susanne, Couto Africa, Marra Vincent, Keleman Krystyna, Dickson Barry J
Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Dr. Bohr-Gasse 3-5, A-1030 Vienna, Austria.
Nature. 2007 Jul 12;448(7150):151-6. doi: 10.1038/nature05954.
Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan.
在模式生物中进行的正向遗传筛选为发育、生理学和病理学的诸多方面提供了重要见解。随着全基因组序列的可得以及RNA介导的基因干扰（RNAi）技术的引入，系统性的反向遗传筛选现在也成为可能。到目前为止，此类全基因组RNAi筛选大多局限于培养细胞以及秀丽隐杆线虫中普遍存在的基因失活。这种强大的方法尚未以组织特异性的方式应用。在此，我们报告了黑腹果蝇RNAi转基因全基因组文库的构建及验证，该文库能够在完整生物体的特定组织中实现基因功能的条件性失活。我们的RNAi转基因由作为反向重复序列克隆的短基因片段组成，并使用二元GAL4/UAS系统进行表达。我们生成了22270个转基因品系，覆盖了果蝇基因组中88%的预测蛋白质编码基因。分子和表型分析表明，这些转基因中的大多数是有功能的。因此，我们的转基因RNAi文库为在果蝇寿命的任何阶段对任何组织中的基因功能进行系统性分析开辟了前景。
