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A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila

Abstract

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.

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Figure 1: A genome-wide transgenic RNAi library.
Figure 2: Efficient and specific gene interference with ubiquitous RNAi.
Figure 3: Tissue-specific RNAi and the enhancing effect of Dicer-2.
Figure 4: RNAi in neurons and muscles.

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Acknowledgements

We thank S. Bicker, J. Buch, M. Garstkiewicz, A. Gruber, D. Hofmann Rodrigues, K. Jäger, S. Krüttner, J. Mayerhofer, D. Muggenhumer, E. Muhr, K. Schernhuber, J. Schluder, A. Schmatz, C. Sturtzel, M. Vinzenz and W. Wolfgang for technical assistance, R. Lehmann for the original hs-hid lines, J. Mummery-Widmer, M. Yamazaki and J. Knoblich for suggesting the use of pnr-GAL4, R. Carthew for initial discussions on the effectiveness of UAS-IR transgenes, and B. Thompson and V. Siegel for helpful comments on the manuscript. F.S. was supported by a long-term postdoctoral fellowship from the Human Frontier Science Program. This work was supported by funds from the Austrian Academy of Sciences (IMBA) and Boehringer Ingelheim GmbH (IMP), and grants from the Austrian Science Fund and the European Union Framework Programme

Author Contributions G.D. established the methodology, and participated in and led the team that constructed the library. K.K. led this team during the finishing stages. D.C. and G.D. performed the bioinformatic analyses, G.D., F.S. and M.F. compiled all the data, which were analysed by G.D., B.J.D. and F.S. K.S. established the enhancing effect of UAS-Dcr-2. B.J.D. conceived and coordinated the project, and wrote the manuscript with input from G.D. and F.S. The remaining authors made major technical contributions to the construction of the library.

The transgenic RNAi library, including detailed information on each line, is available from the VDRC at 〈http://www.vdrc.at〉.

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Correspondence to Barry J. Dickson.

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Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

This file contains Supplementary Table 1 with molecular details of all UAS-IR constructs, including predictions of ON- and OFF-target genes, CAN repeats, and specificity score (s19). Specific details of potential OFF-target genes, including the number of possible 19-mer matches for each UAS-IR construct and each OFF-target gene, are available at http://www.vdrc.at. (XLS 10350 kb)

Supplementary Table 2

This file contains Supplementary Table 2 with all established transgenic RNAi stocks, indicating the linkage of the UAS-IR transgene and its viability and fertility in homozygotes. All lines are in an isogenic w1118 background and have been verified to contain the indicated transgene by genomic PCR. (XLS 1430 kb)

Supplementary Table 3

This file contains Supplementary Table 3 with relative knock-down of target gene mRNA levels. Data are mean ± s.e.m (n = 2). (PDF 18 kb)

Supplementary Table 4

This file contains Supplementary Table 4 with phenotypic scores (0 – 10) for each defect class, for each of the two assays performed with each line crossed to actin5C-GAL4. (XLS 3351 kb)

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Dietzl, G., Chen, D., Schnorrer, F. et al. A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448, 151–156 (2007). https://doi.org/10.1038/nature05954

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