A gene-specific T2A-GAL4 library for Drosophila.
A gene-specific T2A-GAL4 library for Drosophila.
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DOI：
10.7554/elife.35574
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发表时间：
2018-03-22
期刊：
影响因子：
7.7
通讯作者：
Bellen HJ
中科院分区：
文献类型：
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作者：
Lee PT;Zirin J;Kanca O;Lin WW;Schulze KL;Li-Kroeger D;Tao R;Devereaux C;Hu Y;Chung V;Fang Y;He Y;Pan H;Ge M;Zuo Z;Housden BE;Mohr SE;Yamamoto S;Levis RW;Spradling AC;Perrimon N;Bellen HJ
我们产生了约1000个果蝇原种的文库，其中我们在基因的内含子中插入了一个构建体，该构建体允许在内源启动子的控制下表达GAL 4，同时用GAL 4的3'端的多聚腺苷酸化信号阻止转录。这允许许多应用。首先，约90%的必需基因插入会导致严重的功能丧失表型，这是一种有效的基因诱变方法。有趣的是，通过CRISPR工程改造的12/14染色体不携带第二位点致命突变。第二，26/36（70%）的致命插入测试拯救了一个单一的UAS-cDNA构建体。第三，与许多GAL 4插入相关的功能丧失表型可以通过用UAS-翻转酶切除来恢复。第四，GAL 4驱动的UAS-GFP/RFP以高灵敏度报告基因表达的组织和细胞类型特异性。我们报告了数百个以前没有报道过的基因的表达。最后，插入的盒可以用GFP或任何DNA替换。这些股票是评估基因功能的强大资源。确定新发现的基因在体内发挥什么作用是遗传学的重要组成部分。这项任务需要关于每个基因的大量额外信息，例如基因活跃的特定细胞，或者当基因被删除时会发生什么。为了回答这些问题，研究人员需要工具和方法来操纵生物体内的基因。果蝇对这种实验很有用，因为已经有了一个遗传技术工具箱。果蝇的基因编辑允许从动物DNA的任何地方删除或添加小片段遗传信息。另一个工具，称为GAL 4-UAS，是一个用于研究基因活性的两部分系统。GAL 4成分是一种蛋白质，可以打开基因。GAL 4在果蝇细胞中的作用很小，因为它只识别一种叫做UAS的DNA序列。然而，如果一个产生GAL 4的细胞也被改造成含有UAS控制的基因，GAL 4将打开基因。Lee等人使用基因编辑将一小段DNA插入到许多不同的果蝇基因中，其中含有GAL 4序列，然后是“停止”信号。插入使每个基因正常活跃的细胞产生GAL 4，但是-由于停止信号-使原始基因的其余部分失去功能。这有效地删除了每个基因编码的蛋白质，提供了它们通常控制的生物过程的信息。Lee等人继续使用他们的插入方法建立了果蝇基因库。这是一个大约1,000种不同果蝇品系的集合，每种果蝇在单个基因中携带GAL 4/stop组合。该文库允许通过将GAL 4与不同的UAS控制的遗传工具相结合来详细研究集合中的任何基因。例如，引入一个UAS控制的标记将精确定位原始基因在体内的活跃位置。或者，添加UAS控制的人类版本的基因将创造人源化苍蝇，这是研究人类潜在致病基因的宝贵工具。这个果蝇文库是一个资源，为果蝇遗传学提供了新的实验工具。从果蝇身上获得的见解也可以应用于人类等更复杂的动物，特别是因为人类和果蝇之间大约65%的基因是相似的。因此，Lee等人希望这一资源将有助于其他研究人员对许多不同基因在健康和疾病中的作用进行新的阐述。
We generated a library of ~1000 Drosophila stocks in which we inserted a construct in the intron of genes allowing expression of GAL4 under control of endogenous promoters while arresting transcription with a polyadenylation signal 3’ of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36 (70%) of lethal insertions tested are rescued with a single UAS-cDNA construct. Third, loss-of-function phenotypes associated with many GAL4 insertions can be reverted by excision with UAS-flippase. Fourth, GAL4 driven UAS-GFP/RFP reports tissue and cell-type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with GFP or any DNA. These stocks comprise a powerful resource for assessing gene function. Determining what role newly discovered genes play in the body is an important part of genetics. This task requires a lot of extra information about each gene, such as the specific cells where the gene is active, or what happens when the gene is deleted. To answer these questions, researchers need tools and methods to manipulate genes within a living organism. The fruit fly Drosophila is useful for such experiments because a toolbox of genetic techniques is already available. Gene editing in fruit flies allows small pieces of genetic information to be removed from or added to anywhere in the animal’s DNA. Another tool, known as GAL4-UAS, is a two-part system used to study gene activity. The GAL4 component is a protein that switches on genes. GAL4 alone does very little in Drosophila cells because it only recognizes a DNA sequence called UAS. However, if a GAL4-producing cell is also engineered to contain a UAS-controlled gene, GAL4 will switch the gene on. Lee et al. used gene editing to insert a small piece of DNA, containing the GAL4 sequence followed by a ‘stop’ signal, into many different fly genes. The insertion made the cells where each gene was normally active produce GAL4, but – thanks to the stop signal – rendered the rest of the original gene non-functional. This effectively deleted the proteins encoded by each gene, giving information about the biological processes they normally control. Lee et al. went on to use their insertion approach to make a Drosophila genetic library. This is a collection of around 1,000 different strains of fly, each carrying the GAL4/stop combination in a single gene. The library allows any gene in the collection to be studied in detail simply by combining the GAL4 with different UAS-controlled genetic tools. For example, introducing a UAS-controlled marker would pinpoint where in the body the original gene was active. Alternatively, adding UAS-controlled human versions of the gene would create humanized flies, which are a valuable tool to study potential disease-causing genes in humans. This Drosophila library is a resource that contributes new experimental tools to fly genetics. Insights gained from flies can also be applied to more complex animals like humans, especially since around 65% of genes are similar across humans and Drosophila. As such, Lee et al. hope that this resource will help other researchers shed new light on the role of many different genes in health and disease.