A genome-scale shRNA resource for transgenic RNAi in Drosophila

doi:10.1038/NMETH.1592

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A genome-scale shRNA resource for transgenic RNAi in Drosophila

Journal Article•DOI•

A genome-scale shRNA resource for transgenic RNAi in Drosophila

Jian-Quan Ni¹, Rui Zhou¹, Rui Zhou², Benjamin Czech³, Lu Ping Liu¹, Lu Ping Liu², Laura Holderbaum¹, Donghui Yang-Zhou¹, Hye Seok Shim¹, Rong Tao¹, Dominik Handler, Phillip Karpowicz¹, Richard Binari¹, Matthew A. Booker¹, Julius Brennecke, Lizabeth A. Perkins⁴, Gregory J. Hannon³, Norbert Perrimon¹ - Show less +14 more•Institutions (4)

Howard Hughes Medical Institute¹, Sanford-Burnham Institute for Medical Research², Watson School of Biological Sciences³, Harvard University⁴

01 May 2011-Nature Methods (Nature Publishing Group)-Vol. 8, Iss: 5, pp 405-407

TL;DR: It is shown that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis and the progress toward building a genome-wide shRNA resource is described.

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Abstract: Short hairpin RNAs, expressed from microRNA scaffold–containing vectors, efficiently silence gene expression in female germ cells as well as somatic cells in the fly. A genome-wide resource is being developed.

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Journal Article•DOI•

An integrative approach to ortholog prediction for disease-focused and other functional studies

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Yanhui Hu¹, Ian Flockhart¹, Arunachalam Vinayagam¹, Clemens Bergwitz¹, Bonnie Berger, Norbert Perrimon², Norbert Perrimon¹, Stephanie E. Mohr¹ - Show less +4 more•Institutions (2)

Harvard University¹, Howard Hughes Medical Institute²

31 Aug 2011-BMC Bioinformatics

TL;DR: DIOPT as discussed by the authors integrates existing approaches, facilitating rapid identification of orthologs among human, mouse, zebrafish, C. elegans, Drosophila, and S. cerevisiae.

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Abstract: Mapping of orthologous genes among species serves an important role in functional genomics by allowing researchers to develop hypotheses about gene function in one species based on what is known about the functions of orthologs in other species. Several tools for predicting orthologous gene relationships are available. However, these tools can give different results and identification of predicted orthologs is not always straightforward. We report a simple but effective tool, the D rosophila RNAi Screening Center I ntegrative O rtholog P rediction T ool (DIOPT; http://www.flyrnai.org/diopt ), for rapid identification of orthologs. DIOPT integrates existing approaches, facilitating rapid identification of orthologs among human, mouse, zebrafish, C. elegans, Drosophila, and S. cerevisiae. As compared to individual tools, DIOPT shows increased sensitivity with only a modest decrease in specificity. Moreover, the flexibility built into the DIOPT graphical user interface allows researchers with different goals to appropriately 'cast a wide net' or limit results to highest confidence predictions. DIOPT also displays protein and domain alignments, including percent amino acid identity, for predicted ortholog pairs. This helps users identify the most appropriate matches among multiple possible orthologs. To facilitate using model organisms for functional analysis of human disease-associated genes, we used DIOPT to predict high-confidence orthologs of disease genes in Online Mendelian Inheritance in Man (OMIM) and genes in genome-wide association study (GWAS) data sets. The results are accessible through the DIOPT diseases and traits query tool (DIOPT-DIST; http://www.flyrnai.org/diopt-dist ). DIOPT and DIOPT-DIST are useful resources for researchers working with model organisms, especially those who are interested in exploiting model organisms such as Drosophila to study the functions of human disease genes.

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583 citations

Journal Article•DOI•

Highly Improved Gene Targeting by Germline-Specific Cas9 Expression in Drosophila

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Shu Kondo¹, Ryu Ueda¹•Institutions (1)

Graduate University for Advanced Studies¹

01 Nov 2013-Genetics

TL;DR: A simple yet extremely efficient platform for systematic gene targeting by the RNA-guided endonuclease Cas9 in Drosophila, which demonstrates rapid generation of mutants in seven neuropeptide and two microRNA genes in which no mutants have been described.

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Abstract: We report a simple yet extremely efficient platform for systematic gene targeting by the RNA-guided endonuclease Cas9 in Drosophila. The system comprises two transgenic strains: one expressing Cas9 protein from the germline-specific nanos promoter and the other ubiquitously expressing a custom guide RNA (gRNA) that targets a unique site in the genome. The two strains are crossed to form an active Cas9-gRNA complex specifically in germ cells, which cleaves and mutates the target site. We demonstrate rapid generation of mutants in seven neuropeptide and two microRNA genes in which no mutants have been described. Founder animals stably expressing Cas9-gRNA transmitted germline mutations to an average of 60% of their progeny, a dramatic improvement in efficiency over the previous methods based on transient Cas9 expression. Simultaneous cleavage of two sites by co-expression of two gRNAs efficiently induced internal deletion with frequencies of 4.3-23%. Our method is readily scalable to high-throughput gene targeting, thereby accelerating comprehensive functional annotation of the Drosophila genome.

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567 citations

Cites background from "A genome-scale shRNA resource for t..."

...We and others have successfully generated genome-wide collections of transgenic RNA interference lines and distributed them to the community (Leulier et al. 2002; Dietzl et al. 2007; Ni et al. 2011)....
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Journal Article•DOI•

A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine in Flies, Fish, and Mice.

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Fangmiao Sun¹, Jianzhi Zeng¹, Miao Jing², Miao Jing¹, Miao Jing³, Jingheng Zhou⁴, Jiesi Feng¹, Jiesi Feng³, Jiesi Feng², Scott F. Owen⁵, Yichen Luo³, Funing Li⁶, Huan Wang³, Huan Wang¹, Takashi Yamaguchi⁷, Zihao Yong¹, Yijing Gao⁷, Wanling Peng⁶, Lizhao Wang⁸, Siyu Zhang⁸, Jiulin Du⁶, Dayu Lin⁷, Min Xu⁶, Anatol C. Kreitzer⁹, Guohong Cui⁴, Yulong Li³, Yulong Li¹, Yulong Li² - Show less +24 more•Institutions (9)

McGovern Institute for Brain Research¹, Tsinghua University², Peking University³, National Institutes of Health⁴, Gladstone Institutes⁵, Chinese Academy of Sciences⁶, New York University⁷, Shanghai Jiao Tong University⁸, University of California, San Francisco⁹

12 Jul 2018-Cell

TL;DR: The development of genetically encoded GPCR-activation-based-DA (GRABDA) sensors enable spatiotemporally precise measurements of DA dynamics in a variety of model organisms while exhibiting complex behaviors.

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545 citations

Cites background from "A genome-scale shRNA resource for t..."

...UAS-DAT-RNAi (TH01470....
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...FVB(Cg)-Tg(Th-cre)FI172Gsat/ Mmucd MMRRC RRID:MMRRC_031029-UCD Mouse: DAT-IRES-Cre Jackson Laboratory Stock#006660 Zebrafish: albino (slc45a2b4) (The background strain) ZFIN N/A Zebrafish: Tg (elval3: DA1m) This paper N/A Zebrafish: Tg (DAT:TRPV1-TagRFP) Tg; (elval3: DA1m) This paper N/A D. melanogaster: UAS-DA1m/cyo (The line carrying DA1m on the chromosome 2) This paper N/A D. melanogaster: UAS-DA1m/TM2 (The line carrying DA1m on the chromosome 3) This paper N/A D. melanogaster: TH-GAL4 Gift from Yi Rao Unpublished D. melanogaster: C305a-GAL4 Gift from Yi Rao BDSC: 30829 D. melanogaster: 30y-GAL4 Gift from Yi Rao BDSC: 30818 D. melanogaster: DTHFS+/ ple2/TM6B (Cichewicz et al., 2017) Gift from Jay Hirsh N/A D. melanogaster: UAS-DAT-RNAi (Ni et al., 2011) Tsinghua Fly center Tsinghua Fly center: TH01470....
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...melanogaster: UAS-DAT-RNAi (Ni et al., 2011) Tsinghua Fly center Tsinghua Fly center: TH01470....
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...The flies corresponding to figures: Figures 3A–3C UAS-DA1m/cyo; TH-GAL4 (DANs)/TM6B UAS-DA1m-mut/+; TH-GAL4/+ C305a-GAL4 (a’ and b’ Kenyon cells)/UAS-DA1m; DTHFS+/-ple2/+ (WT group) C305a-GAL4/UAS-DA1m; DTHFS+/–ple2 (TH-deficient group) Figures 3D–3K UAS-DA1m/cyo; TH-GAL4/TM6 UAS-DA1m-mut/+; TH-GAL4/+ e4 Cell 174, 481–496.e1–e9, July 12, 2018 Figures 3L–3O UAS-DA1m/cyo; TH-GAL4/TM6B UAS-DA1m/+; TH-GAL4/UAS-DAT-RNAi Figures S5A and S5B UAS-DA1m/cyo; TH-GAL4/TM6B Fig....
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...N) (Ni et al., 2011), from Tsinghua Fly center, Tsinghua University....
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Journal Article•DOI•

The Transgenic RNAi Project at Harvard Medical School: Resources and Validation

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Lizabeth A. Perkins¹, Laura Holderbaum¹, Rong Tao¹, Yanhui Hu¹, Richelle Sopko¹, Kim McCall², Donghui Yang-Zhou¹, Ian Flockhart¹, Richard Binari³, Richard Binari¹, Hye Seok Shim¹, Audrey Miller¹, Amy Housden¹, Marianna Foos¹, Sakara Randkelv¹, Colleen F. Kelley¹, Pema Namgyal¹, Christians Villalta³, Christians Villalta¹, Lu Ping Liu¹, Lu Ping Liu⁴, Xia Jiang⁴, Qiao Huan-Huan⁴, Xia Wang⁴, Asao Fujiyama⁵, Atsushi Toyoda⁵, Kathleen Ayers⁶, Allison Blum³, Allison Blum⁷, Benjamin Czech⁸, Ralph A. Neumüller¹, Dong Yan¹, Amanda Cavallaro³, Karen L. Hibbard³, Donald Hall³, Lynn Cooley⁶, Gregory J. Hannon⁸, Ruth Lehmann⁷, Ruth Lehmann³, Annette L. Parks, Stephanie E. Mohr¹, Ryu Ueda⁵, Shu Kondo¹, Shu Kondo⁵, Jian-Quan Ni¹, Jian-Quan Ni⁴, Norbert Perrimon¹, Norbert Perrimon³ - Show less +44 more•Institutions (8)

Harvard University¹, Boston University², Howard Hughes Medical Institute³, Tsinghua University⁴, National Institute of Genetics⁵, Yale University⁶, New York University⁷, University of Cambridge⁸

01 Jan 2015-Genetics

TL;DR: The various tools developed and the status of the TRiP collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes, are described.

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Abstract: To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China).

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469 citations

Cites background or methods or result from "A genome-scale shRNA resource for t..."

...For shRNA expression we developed the second-generation knockdown vectors, VALIUM20, VALIUM21, and VALIUM22 (Table 1 and Figure S1)(Ni et al. 2011)....
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...Subsequently, we showed that shRNAs containing a 21-bp targeting sequence embedded into a micro-RNA (miR-1) backbone are very effective for gene knockdown in both the germline and soma (Ni et al. 2011)....
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...Either dsRNA or shRNA constructs were generated individually in 96-well plates, or, in the case of shRNAs, they were selected from the shRNA libraries generated in VALIUM20 and VALIUM22 starting from a pool of 83,256 unique shRNA oligonucleotides synthesized on glass slide microarrays (Ni et al. 2011)....
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...In Table 1 and Figure S1we list the most commonly used vectors as well as those not previously reported....
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... The TRiP Valium series vectors C 20 SI L. A. Perkins et al. Figure S1 The TRiP vectors....
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Journal Article•DOI•

Genetic Manipulation of Genes and Cells in the Nervous System of the Fruit Fly

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Koen J. T. Venken¹, Julie H. Simpson², Hugo J. Bellen¹•Institutions (2)

Baylor College of Medicine¹, Howard Hughes Medical Institute²

20 Oct 2011-Neuron

TL;DR: Many of the techniques that help assess the role of specific neurons by labeling, removing, or altering their activity are summarized and an attempt to acquaint the reader with available options and contexts to apply these methods.

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392 citations

Cites background from "A genome-scale shRNA resource for t..."

...See text for details. libraries (Ni et al., 2011). shRNA-mediated RNAi can be directed toward alternative exons and allowed studying the function of alternative splice variants (Shi et al., 2007; Yu et al., 2009b)....
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...libraries (Ni et al., 2011)....
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References

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Journal Article•DOI•

Analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method

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Kenneth J. Livak¹, Thomas D. Schmittgen²•Institutions (2)

Applied Biosystems¹, Washington State University²

01 Dec 2001-Methods

TL;DR: The 2-Delta Delta C(T) method as mentioned in this paper was proposed to analyze the relative changes in gene expression from real-time quantitative PCR experiments, and it has been shown to be useful in the analysis of realtime, quantitative PCR data.

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139,407 citations

Journal Article•DOI•

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

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Georg Dietzl¹, Doris Chen¹, Frank Schnorrer², Kuan-Chung Su¹, Yulia Barinova¹, Michaela Fellner¹, Michaela Fellner², Beate Gasser¹, Kaolin Kinsey², Kaolin Kinsey¹, Silvia Oppel¹, Silvia Oppel², Susanne Scheiblauer¹, Africa Couto², Vincent Marra¹, Krystyna Keleman¹, Krystyna Keleman², Barry J. Dickson¹, Barry J. Dickson² - Show less +15 more•Institutions (2)

Austrian Academy of Sciences¹, Research Institute of Molecular Pathology²

12 Jul 2007-Nature

TL;DR: 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 and opening up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophile lifespan.

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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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2,721 citations

Journal Article•DOI•

Discrete Small RNA-Generating Loci as Master Regulators of Transposon Activity in Drosophila

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Julius Brennecke¹, Alexei A. Aravin¹, Alexander Stark², Monica Dus¹, Manolis Kellis², Ravi Sachidanandam¹, Gregory J. Hannon¹ - Show less +3 more•Institutions (2)

Watson School of Biological Sciences¹, Broad Institute²

23 Mar 2007-Cell

TL;DR: Examination of piwi-interacting RNAs associated with each Drosophila Piwi protein finds that Piwi and Aubergine bind RNAs that are predominantly antisense to transposons, whereas Ago3 complexes contain predominantly sense piRNAs.

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2,303 citations

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Ovarian development in Drosophila melanogaster

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Robert C. King

01 Jan 1970

1,356 citations

Journal Article•DOI•

A Distinct Small RNA Pathway Silences Selfish Genetic Elements in the Germline

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Vasily V. Vagin¹, Alla A. Sigova¹, Chengjian Li¹, Hervé Seitz¹, Vladimir A. Gvozdev, Phillip D. Zamore¹ - Show less +2 more•Institutions (1)

University of Massachusetts Medical School¹

21 Jul 2006-Science

TL;DR: The data suggest that rasiRNAs protect the fly germline through a silencing mechanism distinct from both the miRNA and RNA interference pathways.

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Abstract: In the Drosophila germline, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. Whereas small interfering RNAs (siRNAs) derive from both the sense and antisense strands of their double-stranded RNA precursors, rasiRNAs arise mainly from the antisense strand. rasiRNA production appears not to require Dicer-1, which makes microRNAs (miRNAs), or Dicer-2, which makes siRNAs, and rasiRNAs lack the 2',3' hydroxy termini characteristic of animal siRNA and miRNA. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. Our data suggest that rasiRNAs protect the fly germline through a silencing mechanism distinct from both the miRNA and RNA interference pathways.

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1,279 citations