Breaking Reports: Cell-Based Screens

Catching up on my reading today. On the list:

Asensio et al. (2010) RNAi screen identifies a role for adaptor protein AP-3 in sorting to the regulated secretory pathway. J Cell Biol. 2010 Dec 13;191(6):1173-87. Protocol Notes: Screened in S2 cells using a library of "7,216 sequences that target Drosophila genes conserved to mammals."

Grimberg et al. (2010) Basic-Leucine Zipper Protein Cnc-C is a Substrate and Transcriptional Regulator of the Drosophila 26S Proteasome. Mol Cell Biol. Dec 13. Epub ahead of print. Protocol Notes: Screened the DRSC Transcription Factor library.

Primary Cells

On my desk today? A report that uses RNAi and primary cells to study muscles.

Rui et al. (2010) Sarcomere formation occurs by the assembly of multiple latent protein complexes. PLoS Genet. 6(11):e1001208.

Nominate Genes for TRiP Stock Production

The Trangenic RNAi Project (TRiP) is still taking community nominations for genes to go into the production pipeline for in vivo TRiP RNAi stocks. Nominate!

See the bottom of this page for the contact email for nomination:

http://www.flyrnai.org/TRiP-ACC-OVR.html

Ouch! That's hot.

On my desk today? Report of an in vivo screen for genes involved in heat avoidance.

Neely et al. (2010) A genome-wide Drosophila screen for heat nociception identifies alpha-2-delta-3 as an evolutionarily conserved pain gene. Cell 143(4):628-38. PMID: 21074052

Breaking Reports: Full-Genome RNAi on cell spreading & a Library Evaluation

On my desk today? Catching up on breaking reports from two different groups--one is a full-genome cell-based RNAi screen and the other a comparative report on genome-wide libraries.

D'Ambrosio & Vale (2010) A whole genome RNAi screen of Drosophila S2 cell spreading performed using automated computational image analysis. J Cell Bio 191(3):471-8. PMID: 21041442.

Horn et al. (2010) Design and evaluation of genome-wide libraries for RNA interference screens. Genome Biol. 11(6):R61. PMID 20550664.

Web Wednesday: RNAi Select

Following up on a recent post--breaking paper on dsRNA design--here's the link to the tool reported in the paper.

http://www.rnaiselect.info/dsrna/

Breaking Report: piRNAs

On my desk to read today? A report on the RNA biology of piRNAs, that includes work using dsRNA-based knockdown of piwi in cultured cells.

Haase et al. (2010) Probing the initiation and effector phases of the somatic piRNA pathway in Drosophila. Genes Dev. 2010 Oct 21. [Epub ahead of print]

Breaking Report: Limiting Off-Target Effects

Also on my desk today? An informatics analysis and tool for further limiting potential off-target effects in dsRNA design.

Seinen et al. (2010) RNAi Experiments in D. melanogaster: Solutions to the Overlooked Problem of Off-Targets Shared by Independent dsRNAs. PLoS One. 2010 Oct 1;5(10). pii: e13119.

Breaking Report: DRSC screen for modifiers of Notch transcriptional activity

On my desk today? A report of a screen performed using the DRSC full-genome library.

Mourikis et al. (2010) Modifiers of notch transcriptional activity identified by genome-wide RNAi. BMC Developmental Biology. http://www.biomedcentral.com/1471-213X/10/107

Breaking Report: Myc study points to dHCF

Also on my desk today? Another cell-based RNAi screen report, this one looking at activation of Myc target genes.

Furrer et al. (2010) Drosophila Myc interacts with Host Cell Factor (dHCF) to activate transcription and control growth. J Biol Chem. 2010 Oct 11. [Epub ahead of print]

Breaking Report: Splicing mapk

What's on my desk to read today? A breaking report by Ashton-Beaucage et al. that includes cell-based RNAi screening and in vivo RNAi screening as follow-up.

Ashton-Beaucage et al. (2010) The exon junction complex controls the splicing of MAPK and other long intron-containing transcripts in Drosophila. Cell. 2010 Oct 15;143(2):251-62.

Recent Review: miRNAs & Cancer

What's on my desk to read today? A recent review on miRNAs and cancer.

This review is not directly relevant to using RNAi for screens, of course. But Fig. 4 is a nice summary of tools for disrupting miRNAs. And I like to try to keep up with aspects of RNA biology, as understanding RNA biology is interlinked with exploiting it as a research tool.

Garzon et al. (2010) Targeting microRNAs in cancer: rationale, strategies and challenges. Nature Reviews Drug Discovery 9: 775-789. Available FREE.

Breaking Report: Flight

On my desk today? A new report on the Flight database.

Sims et al. (2010) The flight Drosophila RNAi database: 2010 update. Fly (Austin). 2010 Oct 10;4(4).

Travel Grants for Interdisciplinary Collaborations

Just saw this announced: Burrows Welcome Fund Collaborative Research Travel Grant.

From their website: "This program provides ... support for researchers ... to travel ... to a laboratory to acquire a new research technique, to facilitate/begin collaboration, or to attend a course. Applicants must hold a Ph.D. or are studying for a Ph.D. in mathematics, physics, chemistry, computer science, statistics, or engineering who are interested in investigating research opportunities in the biological sciences or to biologists interested in working with physical scientists, mathematicians, engineers, chemists, statisticians, or computer scientists to incorporate their ideas and approaches to answering biological questions."

SILAC & RNAi

Also on my desk today? A chapter with protocols on combining SILAC-labeling and mass spec with RNAi in Drosophila cultured cells.

Cuomo and Bonaldi (2010) Systems Biology “On-the-Fly”: SILAC-Based Quantitative Proteomics and RNAi Approach in Drosophila melanogaster. Methods Mol Biol. 662:59-78.

Breaking Report: Jak/Stat

What's on my desk to read today? A report that includes screening of a focused set of 67 candidate genes in Kc cells.

Vidal et al. (2010) Negative regulation of Drosophila JAK-STAT signalling by endocytic trafficking. J Cell Sci. 2010 Sep 14. [Epub ahead of print]

New on the DRSC Website

We've just added a map showing empty and control wells on an example plate from the Kinases & Phosphatases set on the sub-libraries web page. For 'the subs' we leave empty wells around the edges to minimize the potential impact of edge-effects on experimental data.

Meeting Announcement

Just received an announcement for the 5th Annual Arthropod Genomics Symposium, June 9 to 12, 2011, Kansas City Marriott on the beautiful Country Club Plaza.

Methods Video

What am I watching today? A new video on fly RNAi methods.

Moser et al. (2010) RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells. J Vis Exp. 2010 Aug 25;(42). pii: 2137. doi: 10.3791/2137.

Just the Facts, Please

We've just updated our Assay Overview page, which describes the instruments available at the DRSC (or at collaborating facilities) for high-content imaging, plate-reader, etc. screens. It's not beautiful design but we hope it gives the information folks need.

Breaking Report: Wound Closure

What's on my desk today? A screen of ~200 in vivo RNAi lines from the VDRC & NIG-Fly collections to identify genes involved in wound closure.

Lesch et al. (2010) A Targeted UAS-RNAi Screen in Drosophila Larvae Identifies Wound Closure Genes Regulating Distinct Cellular Processes. in Genetics.

Presentation on in-cell in situ at HMS

Are you local? The ICCB-Longwood Screening Facility is hosting a workshop on in situ in cells (the Quantigene ViewRNA technology from Affymetrix). See the ICCB-L website for details.

Live Cell Imaging Analysis

What's on my desk today? A report on a new open source software package for analysis of high-throughput live cell images.

Held et al. (2010) CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging. Nature Methods. 7, 747–754.

Quote of the Week: Technology

Quote of of the summer, at this point? It's been a while!

My hypodermic syringe is of the simplest. It consists of a little glass tube, tapering sharply at one end. By drawing in my breath, I fill it with the liquid to be tested; I expel the contents by blowing. Its point is almost as fine as a hair and enables me to regulate the dose to the degree which I want. A cubic millimetre is the usual charge. The injection has to be made at parts that are generally covered with horn. So as not to break the point of my fragile instrument, I prepare the way with a needle, with which I prick the victim at the spot required. I insert the tip of the loaded injector in the hole thus made and I blow. The thing is done in a moment, very neatly and in an orthodox fashion, favourable to delicate experiments. I am delighted with my modest apparatus.

from The Life of the Fly by J.H. Fabre, translated by A. Teixeira de Mattos

(1913, Dodd, Mead & Co., NY)

Another on S2 cells

What's on my desk today? A reprint kindly sent by G. Goshima of the chapter:

Goshima (2010). Assessment of Mitotic Spindle Phenotypes in Drosophila S2 Cells. Methods in Cell Biol. Vol. 97.

Includes discussion of use of RNAi in S2 cells, appropriate controls, variability in the normal cell population, and mitotic mutant phenotypes.

Fly Function Prediction

What's on my desk to read today?

Yan et al. (2010) A Genome-Wide Gene Function Prediction Resource for Drosophila melanogaster. PLoS One. 5(8). pii: e12139. Here's a link to the fly FuncBase website.

Of note: DRSC RNAi screen data sets were used in this study to help evaluate the predictions.

Similar but Different--Comparison of 4 Mammalian RNAi Screens

What's on my desk today? A comparison of the methods used in four mammalian cell RNAi screens investigating HIV-1 host cell factors. Pache et a. (2010) Identifying HIV-1 host cell factors by genome-scale RNAi screening. Methods. doe:10.1016/j.ymeth.2010.07.009.

Pseudomonas Exotoxin & S2 Cells

Also on my desk today? Like today's other post, a study in S2 cells that one could easily imagine leading to a high-throughput screen.

Sharma & Fitzgerald (2010) Pseudomonas Exotoxin kills Drosophila S2 cells via Apoptosis. Toxicon. PMID: 20659495.

Transcriptional response of S2 cells to JH treatment

What's on my desk today?

A report describing the results of a microarray analysis of S2 cells treated with Juvenile Hormone. Several genes up- or down-regulated under these conditions were identified.

Willis et al. (2010) Microarray Analysis of Juvenile Hormone Response in Drosophila melanogaster S2 cells. J Insect Sci. 2010 Jun;10:66. PMID: 2067298.

Genome-Wide Fly RNAi Screen: JAK/STAT

Also on my desk today? Kallio et al. (2010) Eye transformer is a negative regulator of Drosophila JAK/STAT signaling. FASEB Journal fj.10-162784. Screened effects of >16,000 dsRNAs on Jak/Stat signaling using a luciferase reporter in S2 cells.

SID-1 Gives a Boost to Fly RNAi?

What's on my desk today?

Pathak et al. (2010) Evidence that Myosin activity opposes microtubule-based axonal transport of mitochondria. Journal of Neuroscience 30(26):8984-8992.

As described in the report, they use a fly strain expressing C. elegans SID-1 in neurons as a means to boost the RNAi effect in a subsequent primary culture RNAi experiment.

RNAi Biology

What's on my desk today? A summary in Nature Reviews Mol Cell Bio of two recent papers describing an alternative miRNA pathway entitled Dispensable Dicer (A. Heinrichs). It's tough--our understanding of RNA biology is growing quickly--but I like to try to keep on top of the biological phenomena that underly what we exploit for our research.

The two papers discussed in the brief highlight:

• Cheloufi, S. et al. (2010) A Dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature 465, 584–589.
• Cifuentes, D. et al. (2010) A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity. Science 6 May 2010 (doi:10.1126/science.1190809).

Meeting Announcement: Brains in Spain

Just saw this announcement so I thought I'd pass it along: ESF-EMBO's Functional Neurobiology in Minibrains: From Flies to Robots and Back Again will be happening in the autumn. Website indicates that a limited number of travel grants are available for students and early-career researchers. Participation by applicants only and the deadline to apply is in August.

Breaking Report: Host-Pathogen Interactions

What's on my desk today? A just-published, open access report from Akimana et al. (2010) based on a screen performed at the DRSC.

Akimana et al. (2010) Host Factors Required for Modulation of Phagosome Biogenesis and Proliferation of Francisella tularensis within the Cytosol. PLoS One. 2010 Jun 11;5(6):e11025. PMID: 20552012

Visualizing S2 Cells

When I can figure out how to 'visualize' the full report, I'm looking forward to checking out a new video from Buster et al. (2010) Preparation of Drosophila S2 cells for Light Microscopy. J Vis Exp. Jun 3;(40). pii: 1982. doi: 10.3791/1982.

Today & Tomorrow

On my desk today? A review of in vivo RNAi just out from members of our sister-group, the Transgenic RNAi Project (TRiP): Perrimon et al. (2010) in vivo RNAi: Today and Tomorrow. Cold Spring Harb Perspect Biol. PMID: 20534712

Breaking Report: Fly RNAi Methods

On my desk today?

Haley et al. (2010) Vectors and parameters that enhance the efficacy of RNAi-mediated gene disruption in transgenic Drosophila. PNAS. PMID: 20534445.

Breaking Report: Chemical Screen in Fly Cells

What's on my desk this morning? A report from the Eggert lab on a chemical screen performed similar to a cell-based RNAi screen, and including knockdown of Rho in all cells by RNAi.

Castoreno et al. (2010) Small molecules discovered in a pathway screen target the Rho pathway in cytokinesis. Nat. Chem. Biol. 6(6):457-63. PMID 20436488.

(For further reading, see also Eggert et al. (2004) PLoS Biol. Dec;2(12):e379. Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets. Describes the results of RNAi screening performed at the DRSC and a chemical screen.)

Breaking Report: Notch Signaling

What's on my desk today? A new report from Saj et al. that combines results from a high-throughput cell-based RNAi screen with in vivo RNAi-based validation.

Saj A, Arziman Z, Stempfle D, van Belle W, Sauder U, Horn T, Dürrenberger M, Paro R, Boutros M, Merdes G. A combined ex vivo and in vivo RNAi screen for notch regulators in Drosophila reveals an extensive notch interaction network. Dev Cell. 2010 May 18;18(5):862-76. PubMed PMID: 20493818.

Breaking Reports: A small-scale and a genome-wide fly RNAi screen

What's on my desk to read today?

A small, focused Drosophila RNAi screen and another that went genome-wide.

Both papers are related to host cell-pathogen interactions.

Serio et al. (2010) Defining a Core Set of Actin Cytoskeletal Proteins Critical for Actin-Based Motility of Rickettsia. Cell Host Microbe 7(5):388-398.

Valanne et al. (2010) Genome-Wide RNA Interference in Drosophila Cells Identifies G Protein-Coupled Receptor Kinase 2 as a Conserved Regulator of NF-{kappa}B Signaling. J Immunol. 2010 Apr 26. [Epub ahead of print].

Web Wednesday: E-RNAi

A recent report in NAR describes an update to the E-RNAi tool for dsRNA design.

Horn & Boutros (2010) E-RNAi: a web application for the multi-species design of RNAi reagents—2010 update. Nucleic Acids Research. PMID: 20444868.

Web Wednesday: DRSC Gene LookUp ReDesign

We've revamped and added information at our Gene Lookup results page.

Search with a fly gene symbol or other ID, and get a report of what dsRNA amplicons we have for the gene (and if they've hit in screens), what TRiP in vivo RNAi lines we have for the gene, and--scroll to the very bottom--a sense of whether or not the gene is expressed in various cell lines, based on analysis of results from the modENCODE group.

Please check it out! Feedback welcome.

Breaking News: NF-kappa-B Genome-wide Drosophila cell RNAi screen

What's on my desk to read today? A report of a genome-wide fly RNAi screen designed to query the NF kappa B pathway. Includes follow-up studies in zebrafish using morpholinos to knock down gene function in that organism.

Valanne et al. (2010) Genome-Wide RNA Interference in Drosophila Cells Identifies G Protein-Coupled Receptor Kinase 2 as a Conserved Regulator of NF-{kappa}B Signaling. J Immunol. 2010 Apr 26. [Epub ahead of print]. PMID: 20421637.

Web Wednesday: Interlogs

I recently learned about a new online tool and corresponding paper that the authors have used in conjunction with RNAi screen datasets obtained at the DRSC to build interaction networks.

Interlogfinder

Wiles et al. (2010) Building and analyzing protein interactome networks by cross-species comparisons. BMC Systems Biology 4;36.

RNAi Review

Excited to see this week that a PubMed search now pulls up the Mohr (that's me), Bakal & Perrimon review that we've been working on for Annual Reviews of Biochemistry.

Annu Rev Biochem. 2010 Apr 1. [Epub ahead of print]

Genomic Screening with RNAi: Results and Challenges.

Mohr S, Bakal C, Perrimon N.

Abstract: RNA interference (RNAi) is an effective tool for genome-scale, high-throughput analysis of gene function. In the past five years, a number of genome-scale RNAi high-throughput screens (HTSs) have been done in both Drosophila and mammalian cultured cells to study diverse biological processes, including signal transduction, cancer biology, and host cell responses to infection. Results from these screens have led to the identification of new components of these processes and, importantly, have also provided insights into the complexity of biological systems, forcing new and innovative approaches to understanding functional networks in cells. Here, we review the main findings that have emerged from RNAi HTS and discuss technical issues that remain to be improved, in particular the verification of RNAi results and validation of their biological relevance. Furthermore, we discuss the importance of multiplexed and integrated experimental data analysis pipelines to RNAi HTS.

Breaking Report: Genome-Wide

Just back from the fly meeting (including our successful workshop on RNAi in cells and flies--thanks to all participants!). Now it's back to work as usual, with these papers at the top of the reading pile:

A cell-based screen from the Boutros group on innate immunity:

Kuttenkeuler et al. (2010) A large-scale RNAi screen identifies Deaf1 as a regulator of innate immune responses in Drosophila. J Innate Immun. 2010 Feb;2(2):181-94. Epub 2009 Oct 13.

And an in vivo screen from the Penninger group on heart function:

Neely et al. (2010) A global in vivo Drosophila RNAi screen identifies NOT3 as a conserved regulator of heart function. Cell. 2010 Apr 2;141(1):142-53.

Live and In Person!

Myself and co-organizers L. Perkins, M. Boutros and K. Keleman are presenting a workshop in the Friday afternoon workshop session at the 51st ADRC in D.C. this week.

The lineup:

RNAi screening in cells: S. Mohr, D. Sims (FLIGHT), M. Boutros

RNAi screening in vivo: K. Keleman, F. Schnorrer, L. Perkins

Q&A to follow

The emphasis will be on available resources. We have prepared a handout of information about various resources (around the globe and online) for doing Drosophila RNAi in cells and in vivo. If we have left over copies, we will make them available at the registration desk, near the posters, or another obvious and appropriate location after the workshop.

Hope to see you there!

Breaking Report: in vivo RNAi & muscles

Also on my desk today, an in vivo RNAi screen for gene involved in formation and function of muscles. An interesting report--and with beautiful fluorescence images.

Schnorrer et al. (2010) Systematic genetic analysis of muscle morphogenesis and function in Drosophila. Nature. Mar 11;464(7286):287-91. PMID: 2022084

Breaking Report: RNAi on RNA

On my desk today? A report that uses RNAi to study an aspect of RNA biology--in this case, knockdown of specific exosome factors followed by genome-wide analysis of mRNAs is used to gain insight into exosome functions.

Kiss & Andrulis (2010) Genome-wide analysis reveals distinct substrate specificities of Rrp6, Dis3, and core exosome subunits. RNA 16: 781-791. doi: 10.1261/rna.1906710

What's on my desk today? A pile!

• Conrad and Gerlich (2010) Automated microscopy for high-content RNAi screening. In the Journal of Cell Biology.
• Collinet et al. (2010) Systems survey of endocytosis by multiparametric image analysis. In Nature.
• Zhang et al. (2010) Expression in Aneuploid Drosophila S2 Cells. In PLos Biology (open access).

Let's get reading!

Quote of the Week: Tension

... it is the small or minute organisms, or small cellular elements of larger organisms, whose forms will be governed by surface-tension; while the forms of the larger organisms are due to other and non-molecular forces.

from Sir D'Arcy Thompson, On Growth and Form (Abridged Edition, Cambridge University Press, 1961).

Online Protocols Updated at DRSC

Please Note: We've recently updated our dsRNA protocols on-line. View them here.

Take Your Brain to Spain

Recently saw an announcement for this ESF-EMBO symposium: "Functional Neurobiology in Minibrains: From Flies to Robots and Back Again." 17-22 October 2010 in Sant Feliu de Guixols, Spain. Details here.

Quote of the Week

Everything is collage, even genetics.

- from Michael Ondaatje's Divisadero

Bloomington Resources Paper

What's on my desk to read today? A new paper from the Bloomington Drosophila Stock Center (BDSC) that includes information about resources from the TRiP (in vivo RNAi) that are available at the BDSC:

Cook et al. (2010) New research resources at the Bloomington Drosophila Stock Center. Fly. 4(1):1-4.

Breaking Report: Dissecting Signaling with RNAi

What's on my desk today? A newly published report based on extensive double knockdown RNAi screening that made use of DRSC resources.

Nir et al. (2010) Inference of RhoGAP/GTPase regulation using single-cell morphological data from a combinatorial RNAi screen. in Genome Research.

Breaking Report: in vivo rescue

What's on my desk this morning? Another report on RNAi rescue in Drosophila:

Langer et al. (2010) In Vivo RNAi Rescue in Drosophila melanogaster with Genomic Transgenes from Drosophila pseudoobscura. PLoS One. 5(1):e8928. PubMed PMID: 20126626.

Related posts:

Tissue-Specific RNAi Rescue in Drosophila

Cross-Species RNAi Rescue in Cultured Drosophila Cells

Quote of the Week

Enjoying insects is much the same whether you do it in your own garden or in a distant country. The rule is to watch, and interfere as little as possible.

- H. Oldroyd in The Insects in Your Garden (1976, Penguin Books Ltd; England)

Meeting Announcement: Arthropod Genomics

Just received an announcement about the 4th Annual Arthropod Genomics Symposium, Arthropod Genomics: New Approaches and Outcomes. June 10-13 in Kansas City, MO.

Breaking Report: Fly RNAi & Huntington Disease Model

What's on my desk today?

A report that includes data from a genome-wide screen performed at the DRSC:

Zhang et al. (2010) A Genome-wide RNAi Screen for Modifiers of Aggregates Formation by Mutant Huntingtin in Drosophila. Genetics. 2010 Jan 25. [Epub ahead of print] PubMed PMID: 20100940.

The paper includes a note added during preparation comparing these results with results of a similar study by Doumanis et al. (2009) [blogged about here last fall ], as well as comparison with similar work in C. elegans.

Recent Review: RNAi Routes to Studying Aging

Another paper on my desk today--a mini-review on using RNAi screens, such as in C. elegans and in vivo in flies, to study aging.

Minois et al (2010). RNA Interference in Ageing Research - A Mini-Review. Gerontology. PubMed PMID: 20090308.

Related Links (resources for in vivo RNAi in Drosophila):

Vienna Drosophila RNAi Center (VDRC)

NIG-BIO Japan RNAi Fly Stocks

Trangenic RNAi Project (TRiP) at Harvard Med School

Trendy Science? Acai Berries & Fruit Flies

Even Drosophila research is being influenced by the trends--here's a paper on the effects of acai berries in the context of a high-fat diet. Sun et al. (2010) Açai Palm Fruit (Euterpe oleracea Mart.) Pulp Improves Survival of Flies on a High Fat Diet. Exp Gerontol. PubMed PMID: 20080168.

Breaking Report: in vivo RNAi screen on obesity

What's on my desk today? (And okay, I admit, it has been on my desk for several days ... busy times):

Pospisilik et al (2010). Drosophila genome-wide obesity screen reveals hedgehog as a determinant of brown versus white adipose cell fate. Cell. Jan 8;140(1):148-60. PubMed PMID: 20074523.

Breaking Report: Tiling Arrays to Deconvolve shRNA Pools

What am I reading today? Report of a technical development in the 'mammalian world' of RNAi screening.

Boettcher et al. (2010) Decoding pooled RNAi screens by means of barcode tiling arrays. BMC Genomics. 2010 Jan 5;11(1):7.