Web Wednesday: GenomeRNAi

Todays Web Wednesday featuer: GenomeRNAi, an effort from the Boutros group at DKFZ that reports an update in the most recent database issue of NAR:

Gilsdorf et al. (2009) GenomeRNAi: a database for cell-based RNAi phenotypes. 2009 update. Nucleic Acids Res. doi: doi:10.1093/nar/gkp1038.

Database issue of NAR 2009 (free online access)

GenomeRNAi

And check out their "List all Screens" for what I assume is a summary of the screens that are searchable in the database. Included among the screens in Drosophila and human cells listed are many published screens based on data obtained at the DRSC. Looks like a good place to go to see what's out there from many groups.

RNAi in 30 Insect Species and Counting

On my desk today? A recently e-published review by Xavier Belles in the upcoming 2010 edition of Annual Reviews in Entomology, Beyond Drosophila: RNAi in vivo and functional genomics in insects.

Quote of the Week: Contradiction

The female germ cell embodies a most profound contradiction. It must be totipotent, containing within itself all the information needed for the development of a new individual. But it must also be highly differentiated in order to carry out the complex functions required of it.

- Anne McLaren, in Germ Cells and Soma: A New Look at an Old Problem (Yale University Press, New Haven and London, 1981)

Breaking Report: Metazoan Secretory Pathway

What's on my desk to read today (besides the post-holiday pileup of emails)? A recent report based on a screen performed at the DRSC: Wendler et al. (2009) A genome-wide RNA interference screen identifies two novel components of the metzoan secretory pathway. EMBO Journal. Protocols notes: Luciferase assay readout (stable S2 cell line with the reporter construct), DRSC genome-wide library, follow up in human cells with siRNAs.

Local? Imaging Workshops

Local to HMS? Check out upcoming workshops offered by the imaging center. Training on image analysis software and more.

Web Wednesday (well, Thurs)--Pathway Projector

Just learned about Pathway Projector, which describes itself as a zoomable interface for systems biology. Kono et al. (2009) Pathway Projector: Web-Based Zoomable Pathway Browser Using KEGG Atlas and Google Maps API. PLoSOne (free on-line access).

Breaking Report: JNK & Immunity Screen

What's on my desk today?

A report from Bond & Foley (2009) A Quantitative RNAi Screen for JNK Modifiers Identifies Pvr as a Novel Regulator of Drosophila Immune Signaling. PLoS Pathogen (Open Access).

From their abstract, "Our data illuminate a poorly understood arm of a critical and evolutionarily conserved innate immune response." Protocol Notes: Screened S2 cells; library in part from OpenBio; "in-cell western" approach compared phospho-JNK to Actin levels; 15 and 60 minute timepoints.

Quote of the Week: Halloween Horrors

The name 'coffin-fly' has been applied to Conicera tibiais of the family Phoridae, because this fly is able to maintain itself through many successive generations in coffined bodies that have been interred for a year or more. The fact has long been known, but even today no one really knows how the fly gets there. It seems unlikely, for a variety of reasons, that the eggs are laid on the body before burial, and so either the adult fly or the larva must find its way down through several feet of soil. This seems a difficult feat when we think of its being performed by each larva or each adult fly, but, as in many problems to do with animals, time and continuity make most things possible.

from The Natural History of Flies by Harold Oldroyd

Breaking Report: in vivo RNAi to look at cell survival in the fly wing

What am I reading today? A new report from Umemori et al. (2009) RNAi-Mediated Knockdown Showing Impaired Cell Survival in Drosophila Wing Imaginal Disc. Gene Regulation and Systems Biology 2009:3 1-10.

Breaking Report: Allele-Specific siRNAs

What's on my desk today? A report with relevance to RNAi reagent design and treatment of genetic diseases: Huang et al. (2009) Profiling of mismatch discrimination in RNAi enabled rational design of allele-specific siRNAs. NAR. doi:10.1093/nar/gkp835

Quote of the Week: Attachment

Your garden is an insect zoo, where you can go at any time of the year and see what the insects are doing. Some of them come and go, while others stay in the same place all their lives. ... [Most] are not very attractive, like the plant bugs and beetles. This does not mean that they are not interesting, and if you ... look at them every day, you may become quite attached to them.

From, The Insects in Your Garden by Harold Oldroyd

Breaking Reports: Primary Cell RNAi Protocol & Mitochondrial Screen

Bai et al. (2009) Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening. Nat Protoc. 4(10):1502-12.

Jiang et al. (2009) Genome-wide RNAi screen identifies Letm1 as a mitochondrial Ca2+/H+ antiporter. Science. 326(5949):144-7. And see comment: Science 326(5949):57-8.

Demo: Whole-Slide Imaging

The DRSC is hosting a demo instrument for whole-slide imaging. If you're local to the Boston area and interested, please contact me -or- drop by NRB 350 on Mon. Oct. 5 for an introduction to the technology (10-11 AM) and a related talk (11 AM - noon).

Quote of the Week: Spirals

Of true organic spirals we have no lack. We think at once of horns of ruminants, and of still more exquisitely beautiful molluscan shells ... Closey related spirals may be traced in the florets of a sunflower; a true spiral, though not, by the way, so easy of investigation, is seen in the outline of a clodiform leaf; and yet again, we can recognise typical though transitory spirals in a lock of hair, in a staple of wool, in the coil of an elephant's trunk, in the 'circling spirals' of a snake, in the coils of a cuttle-fish's arm, or of a monkey's or a chameleon's tail.

Among such forms as these, and the many others which we might easily add to them, it is obvious that we have to do with things which, though mathematically similar, are biologically speaking fundamentally different; and not only are they biologically remote, but they are also physically different, in regard to the causes to which they are severally due.

Sir D'Arcy Thompson, On Growth and Form

Breaking Report: Huntingtin Aggregation Screen

On my desk today? Doumanis et al. (2009) RNAi Screening in Drosophila Cells Identifies New Modifiers of Mutant Huntingtin Aggregation. PLoS One. 4(9)37275. Free on-line access. Authors report a primary screen in BG2 cells stably expressing a poly-Q EGFP thatform aggregates with a library they generated (about 7200 genes). Follow-up includes in vivo RNAi in fly eye and brain.

Breaking Report: Making dsRNA Vectors

Making dsRNA constructs in vectors? Hauge and colleagues report a one-tube, ligation-independent cloning method. If you're making your own constructs for in vitro or in vivo work, might be worth taking a look at their strategy.

Hauge et al. (2009) Single tube, high throughput cloning of inverted repeat constructs for double-stranded RNA expression. PLoS One. 4(9):e7205.

Breaking Report: Transmembrane Proteins in Embryogenesis

What's on my desk to read today? Zuniga et al. (2009) Genes encoding novel secreted and transmembrane proteins are temporally and spatially regulated during Drosophila melanogaster embryogenesis. BMC Bio. 7(1):61. Why? Pretty pictures! And interesting science.

DRSC Website

We are updating the DRSC website. Please contact us if you have problems accessing the website or the database on or after Mon. Sept. 28th. Thank you.

Quote of the Week

Does there not exist a high ridge where the mountainside of “scientific” knowledge joins the opposite slope of “artistic” imagination?

Vladimir Nabokov (writer and lepidopterist)

New on the DRSC Bookshelf

Two new things on the DRSC bookshelves these days. One practical. One just for fun.

(1) The Practical -- we have compiled a set of protocol & methods papers from the DRSC and other groups and organized them into a three-ring binder, so that people can take a look through and quickly find relevant protocols for cell culture, assays, transfection and so on. We have a digital folder of the PDFs as well.

(2) The Fun -- on my office bookshelf is a copy of the 2009 release Pictorial Webster's: A Visual Dictionary of Curiosities by John M. Carrera (Chronicle Books, San Francisco, CA, USA). Carrera compiled the images largely from 19th century dictionaries. The result is a quirky and interesting compendium of small engravings, many with the same kind of look I am used to from seeing (and appreciating) engraved and hand-drawn figures in older science texts and journals.

Long Life

What I'm reading today? On aging and flies:

Copeland et al. (2009) Extension of Drosophila life span by RNAi of the mitochondrial respiratory chain. Current Biology. 19, 1-8.

Preview: Home

We're planning to update the DRSC home page so it functions more as a "table of contents" and less like text to read. Screenshot of the revised shown here. Feedback welcome. I expect the change will launch before the end of the month.

Hey, Buddy!

What I'm reading today?

A report from Drinnenberg et al. in which they describe introducting RNAi-related genes from other yeast species into the budding yeast S. cerevisiae, successfully making RNAi work in that species.

Drinnenberg et al (2009). RNAi in Budding Yeast. Science (Sept. e-pub ahead of print). PubMed ID: 19745116

Silkworm RNAi

What's on my desk today? A report from Fujita et al. (2009) describing a vector-based approach to RNAi knockdown in a cell line from the silkworm Bombyx mori.

Fujita et al. (2009) DNA Vector-Based RNA Interference in Cell Lines Derived from Bombyx mori. Biosci. Biotechnol. Biochem. Pubmed ID 19734648.

Home

In the works at the DRSC--a change to our home page. Stay tuned. And suggestions welcome.

Quote of the Week: Ready or Not

There is a widespread view that scientific discoveries are more or less inevitable, and that it makes little difference whether or not a particular individual makes a discovery at a given time: if the time is not ripe for it, it will not be understood and will have little or no effect on future events; if the time is ripe, then someone else will soon make the discovery anyhow.

The history of Mendelism is one of the often-cited examples here. According to this interpretation, Mendel's paper was not understood in 1866 because the time was not ripe ... To me, this account seems greatly oversimplified--though it must be admitted that the development of the subject would probably have been much the same, even to the dates, if Mendel's paper had never been written.

It it true that the paper was ahead of its time, but it was not difficult to understand, and it seems unlikely that it would have remained unappreciated for so long if it had appeared in a less obscure journal, or if Mendel himself had published the further cases that he reported to his letters to Nageli.

A History of Genetics (1965) by A.H. Sturtevant, Cold Spring Harbor Laboratory Press.

To the Rescue!

What's on my desk today? A report co-authored by the DRSC's own Matt Booker,

Kondo et al. (2009) Cross-species RNAi Rescue Platform in Drosophila melanogaster. Genetics. PMID: 1972085

An accompanying web tool and resources are available through the DRSC.

Quote of the Week: Pseudo-Obscure

There seemed to be no end to the tricks that [Drosophila] pseudoobscura could do: one stock had odd sex ratios, another diplayed what seemed to be maternal inheritance, and so on. ... Pseudoobscura was so full of new tricks because it was an undomesticated, wild organism that had not yet been stripped of its natural genetic diversity, as melanogaster had long since been.

In, Lords of the Fly (1994) by R.E. Kohler, University of Chicago Press (Chicago, IL). pg. 260.

Breaking Report: RNAi Screen Analysis

Recommended reading:

Birmingham et al. (2009) Statistical methods for analysis of high-throughput RNA interference screens. Nat Methods. 2009 Aug;6(8):569-75.

PubMed PMID: 19644458.

Breaking Report: RNAi Saves Chocolate!?

Caribe dos Santos et al. (2009) dsRNA-induced gene silencing in Moniliophthora perniciosa, the causal agent of witches' broom disease of cacao. In Fungal Genetics & Biology.

Breaking Report: RNA Biology & Immunity

On my desk today:

Gruber et al (2009) Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation. In Cell.

Sabin et al (2009) Ars2 regulates both miRNA- and siRNA- dependent silencing and suppresses RNA virus infection in Drosophila. In Cell.

With accompanying comment.

Quote of the Week: Self-Deception

For it is easy in experimentation to be deceived, and to think one has seen and discovered what we desire to see and discover.

Luigi Galvani, as quoted in in The Ten Most Beautiful Experiments by George Johnson (2008, Vintage Books).

Grant Opportunity for Mammalian RNAi

Thermo recently announced a grant opportunity for mammalian RNAi screening.

Quote of the Week: Diversity in Paradise

In the 1950s, when D. Elmo Hardy began describing the fantastic diversity of native Drosophila, he realized that a marvelous natural evolutionary experiment was occurring in the island forests. It was as if an earlier geneticist had locked specimens of a close relative of Drosophila melanogaster in a number of extremely large test tubes and allowed them to evolve for thousands or even millions of years ... In 1963, the Hawaiian Drosophila Project began diciphering the genetics and evolution of this amazing group of flies. Over 800 species of Drosophilidae in five genera have been recorded in the islands. This is nearly one-third of the total number of known drosophilid species in the world. Their diversity of form, ecology, and behavior far exceeds the range of known variations in the group outside of Hawai'i.

From Hawaiian Insects and Their Kin (1992) by F.G. Howarth & W. P. Mull. University of Hawaii Press (Honolulu, HI). pg.22.

Genetic Mapping

The Bloomington Drosophila Stock Center has announced availability of a Deficiency Kit. A great resource for the fly community!

East Coast Academic Screening Meeting

Are You Local to the East Coat USA & at and Academic Institution? Interested in drug discovery screens? Mark your calendar: East Coast Academic Screening Meeting on Sept.1, 2009 in Cambridge, MA.

RNAi & Cancer Study

Local to Boston? Please check out information about the newly formed Dana Farber/Harvard Cancer Center (DF/HCC) RNAi Core, which brings together local experts, resources and facilities to better serve the DF/HCC cancer research community.

Breaking Report: Fly Model of Leukemia

On my desk today? Osman et al. (2009) A Drosophila model identifies calpains as modulators of the human leukemogenic fusion protein AML1-ETO. In PNAS. PubMed ID 19581587. The authors report a model of AML1-ETO-induced leukemia. Includes results of an in vivo screen using the collection of the National Institute of Genetics (of Japan, I assume). See LINK for information about that fly stock collection.

Quote of the Week

Attempting to describe the relationship between a gene and its related phenotype is fraught with hazards.

From, X-Linked Traits: A Catalog of Loci in Nonhuman Mammals (1990) by James R. Miller (Cambridge University Press).

Web Wednesday: "Personal" Projects

Perhaps as the Church lab is in the same department as the DRSC, I've gotten used to seeing the letters PGP and thinking "Personal Genome Project" (see this link). So maybe you can understand that a quizzical look crossed my face when I saw "DPGP" and thought "Drosophila Personal Genome Project"? Really? Of course, it turns out the DPGP stands for Drosophila Population Genomics Project. But the goals are not so entirely unrelated. At least part of the DPGP plan is to sequence 50 different D. melanogaster genomes, towards understanding more about variation in a population. Visit the site to learn about the project and its goals.

Breaking Report: the modENCODE Project

Genome sequencing is one thing; understanding the sequence data is quite another. In Celniker et al. (2009) Unlocking the secrets of the genome. Nature 459: 927-930, the authors describe the NHGRI-funded modENCODE project to improve annotation of the C. elegans and Drosophila genomes. The expectation is that the data and resources generated in the consortium project will have far-reaching effects. Community input is actively sought for specific sub-projects.

Meeting Announcement: RNAi Europe

Abstract deadline for posters is Aug. 7th for the upcoming fall RNAi Europe meeting to be held in Berlin, Germany by Select Biosciences.

Breaking Report: Wnt Signaling

What's on my desk to read today? Kategaya et al. (2009) Bili Inhibits Wnt/β-Catenin Signaling by Regulating the Recruitment of Axin to LRP6. Free full text from Plos One.

Protocol Notes: Screened Clone 8 Cells (described in DasGupta R, Kaykas A, Moon RT, Perrimon N. Functional genomic analysis of the Wnt-wingless signaling pathway. Science. 2005 May 6;308(5723):826-33). Factors in defining what hits to follow up on included a look at potential off-target effects and comparison to hits identified in previous screen (the authors report that Bili was a weak or non-hit in screens in the DRSC database, suggesting specificity to the Wnt pathway). Follow-up tests included over-expression of Bili in Drosophila S2R+ and Clone 8 cells; test of shRNA directed against Bili in Drosophila embryos; tests in Zebrafish (with morpholinos); and tests in human cells (with siRNAs).

[Drawing above, very rough interpretation of part of their Figure 2.]

Quote of the Week: Independence Day

Independent assortment the random distribution of the gametes of genes located on different chromosomes. Thus, and individual of genotype Aa Bb will produce equal numbers of four types of gametes: AB, Ab, aB, and ab. See Mendel's laws.

Independent probabilities in a group of events, the occurrence of any one event having no influence on the probability of any other event. For example, the orientation of one pair of homologous chromosomes on the first meiotic metaphase plate does not influence the orientation of any other pair of homologs. See independent assortment.

From, A Dictionary of Genetics (6th Edition), R.C. King & W. D. Stansfield (2002: Oxford University Press, Inc., New York, NY).

Web Wednesday: RNAiCut

Today's Web Wednesday feature is the RNAiCut on-line tool, which--roughly speaking--lets you use available information (such as protein-protein interaction data) to 'clean up' a list of hits from a primary screen. 

See also the publication from Kaplow et al. (2009) RNAiCut: automated detection of significant genes from functional genomic screens in Nature Methods. PubMed ID 19564846.

Damaged? Survive!

What's on my desk to read today?  

A report from Ravi et al. (2009) A Network of Conserved Damage Survival Pathways Revealed by a Genomic RNAi Screen.  Free full text from PLoS Genetics. 

Protocol Notes: KC167 cells; full-genome screen; CellTitreGlow assay for viability as read-out. Screen data obtained at the DRSC.

Presidential Smackdown

The New York Times asks "What has 132 Rooms and Flies?"  And answers: the White House. Video clip also on YouTube.

Web Wednesday: FlyMine 17.0

FlyMine recently released their version 17.0.  View release notes here.

Breaking Reports: Bacterial and Viral Invaders and RNAi

What's on my desk to read today?

Cronin et al. (2009) Genome-wide RNAi screen identifies genes involved in intestinal pathogenic bacterial infection. Science. Reports the results of an in vivo screen after infection with the gut pathogen, Serratia marcescens.

Berry et al. (2009) Viral Suppressors of RNA Silencing Hinder Exogenous and Endogenous Small RNA Pathways in Drosophila. Free full text from PLoS One.

Quote of the Week: Mosquiticidal Tendencies

Invariably, advancements are accompanied by the coinage of new terms such as mosquiticidal. It joins a long series of terms including insecticide, nematicide, fungicide, larvicide, adulticide, entomicide, and even homicide, which may be convenient in indicating toxicity to or death of a target. ... The value and appropriateness of such terms remain to be seen. Certainly, in recognition of the current perception of the specificity of these agents [i.e. bacteria that kill mosquitoes or black flies], and their value, they should not be termed biocides.

in Bacterial Control of Mosquitoes & Black Flies, H. de Barjac & D.J. Sutherland, Editors. Rutgers University Press (1990; New Brunswick, NY).

Repeat, Repeat, Repeat

What's on my desk today? A report with advice on the number of replicates necessary to limit false negative and false positive results in RNAi screens--in terms of both the number of negative control wells on each plate and the total number of replicate assay plates to test in primary and secondary screening.

X.D. Zhang & J.F. Heyse (2009) Determination of sample size in genome-scale RNAi screens. Bioinformatics. 25(7):841-844.

First impressions? Their conclusions lead to advice that seems sound, practical and achievable, with different numbers of replicates suggested depending on the experimental goals.

All that and the American Wandering Spider, too

Seeing an announcement for CSHL Press's Emerging Model Organisms: A Laboratory Manual, Volume 1 (link) has made me consider adding a new feature to the blog, "Books I wish I Owned." But would just owning this book be enough? Glancing at the list of emerging models, I am taken by two things. First, I was amused to see Dictyostelium discoideum on the table of contents. PubMed records on Dicty go back as far as the 1930s. Poor Dicty, still "emerging" after all these years! And second, would someone please set up a tank of comb jellies (Ctenophora), a colony of snails (Ilyanassa), or even a small terrarium of moss (Physcomitrella patens) in my office? These sound like fun critters to have around.

Breaking Report: Apoptosis

Also on my desk today? Chew et al. (2009) Genome-wide silencing in Drosophila captures conserved apoptotic effectors. Nature. Primary screen in triplicate with S2R+ cells and Ambion library. Follow-up included time-lapse microscopy and siRNA in human cells.

Breaking Report: RNAi Rescue

What's on my desk today? Schulz et al. (2009) A novel method for tissue-specific RNAi rescue in Drosophila. Nucleic Acids Research. Free on-line access. The authors make use of the degeneracy of the genetic code to create rescue constructs that evade RNAi knockdown in cells or whole animals.

Breaking Report: Transgenic RNAi Flies for Neurogenetics

What's on my desk today? A report from our sister group, the Transgenic RNAi Project (TRiP), which has been funded by HHMI and NIH to produce a large-scale collection of flies for in vivo RNAi.

Ni et al. (2009) A Drosophila Resource of Transgenic RNAi Lines for Neurogenetics. Genetics. PubMed ID 19487563.

Related Links:

TRiP web pages

TRiP toolbox at Bloomington

TRiP flies at Bloomington

VDRC

'Omics & the Wnt Pathway

What's on my desk today? A pair of research reports and an accompanying opinion piece published last fall (November 2008) from R. Moon's lab. The opinion piece emphasizes the power of integrating results obtained via multiple high-throughput strategies. The research reports present examples of these types of integrated studies, with a focus on Wnt/beta-Catenin signaling.

Major et al. (2008) New regulators of Wnt/beta-Catenin signaling revealed by integrative molecular screening. Science Signaling 1:ra12.

James et al. (2008) Bruton's Tyrosine Kinase revealed as a negative regulator of Wnt-beta-Catenin signaling. Science Signaling 2:ra25.

Major & Moon (2008) "Omic" risk assessment. Science Signaling 2:eg7.

Boola Boola

About a year ago, James Rothman and Lars Branden from Yale visited the DRSC. As part of a larger tour they took of similar centers. As planned, they have now opened the doors to the Yale Center for High Throughput Cell Biology. Best wishes for success!

Cell Cyle & Fly RNAi: Methods

Two new methods papers relevant to fly RNAi are out in volume 545 of the Methods in Molecular Biology series from Humana Press (2009). Bettencourt-Dias & Goshima, RNAi in Drosophila S2 cells as a tool for studying cell cycle progression. And Pereira et al. Dissecting mitosis with laser microsurgery and RNAi in Drosophila cells.

Alas, my computer connection conspires to prevent me from taking a look at these this morning. But based on the titles alone, look like they could be of interest to those of you planning Drosophila (or other) RNAi screens that will use a plate-reader, FACS or imaging to address a cell cycle-related question.

Related Links: A few of the sub-categories we list on the DRSC's Completed Screens, By Topic publications list report the results of RNAi screens aimed at learning about cell death, division and related topics.

Quote of the Week: Making Friends

In rural sections of the southern United States, many people believe that if one removes a fly from a spider's web he will gain a friend.

from Wonders of the Fly World, Sigmund A. Lane (1970) Dodd, Mead & Co. New York.

Breaking Reports: Tools for Fly Recombineering

Following up on a Web Wednesday post, the paper from Venken et al. is now out in Nature Methods. Along with another report on fly tools from Ejsmont et al. in the same issue.

Quote of the Week: Beach Colors

The insect-species confined to the sea-coasts, as every collector knows, are often brassy or lurid.

from Charles Darwin's The Origin of Species.

Web Wednesday: Image Analysis

Today's Web Wednesday feature, CellProfiler. 

It's a freely available, open-source software suite for high-throughput image analysis. 

Associated Carpenter et al. (2006) paper at Genome Biology.

Breaking Report: Comparing DRSC Screen Data to PPI Data

What's on my desk today? 

Wang, Tu & Sun (2009) A network-based integrative approach to prioritize reliable hits from multiple genome-wide RNAi screens in Drosophila. BMC Genomics, 10:220.

Why this paper? 

Among other reasons, as it's based on results of 24 screens performed at the DRSC (full citations of these and more here), which the authors downloaded from our openly accessible flyrnai.org website and database. It's great to see that our data sharing policies are facilitating this type of work. 

Also, at the DRSC we are interested in computational and experimental approaches that lead to integration of large datasets. It's becoming clear that analysis of integrated results can improve our understanding and interpretation of individual results. Indeed, Wang et al. conclude in their study that integrating the RNAi results with protein-protein interaction data informs the understanding of false positive and false negative results.

Quote of the Week

If genius is the ability to reduce the complicated to the simple, then the study of p53 makes fools of us all.

Voudsden & Prives (2009) in the review, Blinded by the Light: the Growing Complexity of p53. Cell 137(3):413-431

Meeting Announcement: Arthropod Genomics

Just saw an announcement for the 3rd Annual Arthropod Genomics Symposium: Frontiers in Arthropod Genomics. Registration & Poster abstract deadline is May 15, 2009. From ticks to aphids, flies to mosquitoes--looks like an interesting line-up.

Breaking Report: Tissue-Specific RNA Preps

What am I reading today? A report on tissue-specific isolation of RNA from whole flies. Sounds like a method that could be quite useful as part of in vivo follow-up to cell-based screens.

Miller MR, Robinson KJ, Cleary MD, Doe CQ (2009) TU-tagging: cell type-specific RNA isolation from intact complex tissues. Nat Methods. May 10. PMID: 19430475.

Related post: Report of Single-Cell RNAseq.

Quote of the Week: Diversity in Development

If morphological diversity is all about development, and development results from genetic regulatory programs, then is the evolution of diversity directly related to the evolution of genetic regulatory programs? Simply put, yes. But to understand how diversity evolves, we must first understand the genetic regulatory mechanisms that operate in development. In other words, what is the genetic toolkit of development and how does it operate to build animals?

In, From DNA to Diversity

Carroll, Grenier and Weatherbee (2001, Blackwell Science; p.13).

Breaking Report: Divisions Gone Wrong

What's on my desk today? A paper on a Drosophila kinase RNAi screen and a very recent report that extends the results to a specific topic in human disease.

Bettencourt-Dias et al. (2004) Genome-wide survey of protein kinases required for cell cycle progression. Nature 432:980-987. At PubMed.

Wang et al. (2009) Association of genetic variation in mitotic kinases with breast cancer risk. Breast Cancer Res. Treat. DOI 10.1007/s10549-009-0404-3. At PubMed.

Related Links:

DRSC Kinase & Phosphatase Sub-Library (on- or off-site screening).

Transgenic RNAi Project (TRiP) stock list (click to expand the current list, download the xls file, open in Excel, and see Column H on the spreadsheet to view which hairpin lines target kinase-encoding genes).

Web Wednesday: On-Line Forums for Screen Imaging

For today's Web Wednesday, I'm going to share a link to the newly-created Opera High Content Imaging google group, which was co-founded by a colleague of ours in the HMS Department of Genetics. 

Their stated purpose is "for the users of the Opera platform to exchange tips and tricks when dealing with the Instrument," as well as to exchange Acapella scripts. From the Opera google group page (or this link) you can also get to an on-line group for high-content imaging more generally.

Make Way for Ducklings

It's not all research, all the time here at the DRSC. Thanks to all who joined us for spring fun on Friday.

At HMS or Affiliated? Proteomics Nanocourse

An upcoming half-day course on proteomics offered by Harvard Catalyst might be of interest. Check it out if you're in the area. May 18th 1 to 5 pm at the BIDMC. Registration is required.

Quote of the Week: Flu & Flying

What I recommend people [in airplanes] do is turn the overhead air releaser on to a low stream and position it so the flow goes in front of your face. That will increase the ventilation, at minimal, create a turbulence of air in front of your face. If a particle is coming by, that might be enough to push it out of the way.

M. Gendreau, Tufts University & Lahey Clinic

in When Flu Flies (Newsweek) April 27, 2009

Flies & Flu

With today's news on my mind, I searched for "Drosophila" and "flu" today at PubMed and that led me to: Hao et al. (2008) Drosophila RNAi screen indentifies host genes important for influenza virus replication. Nature 454:890-893. Protocol Notes: Screened in D-Mel2 cells with Ambion dsRNAs. Follow-up in mammalian cells using siRNAs.

Web Wednesday--P[acman] BAC fly resource

Recently learned about a new resource, Drosophila BAC libraries in a vector system for recombineering and transgenesis. Information on-line. Rumor has it a paper by Venken et al. is in press at Nature Methods.

Learn more about the resource on-line here:

http://www.pacmanfly.org/

Breaking Report: A Look at Centromeres

What am I reading today? Another paper that reports results from a genome-wide study performed at the DRSC: Erhardt et al. (2008) Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation. JCB 183(5):805-818.

The authors describe the results of a full-genome screen for factors that disrupt the normal localization of CENP-A (CID). Protocol Notes: A fluorescence image-based screen assay in Kc167 cells. The authors provide a nicely detailed description of the screening protocol, including info on image analysis, in the Materials & Methods section. Follow-up includes time-lapse and additional imaging in S2 cells and fly embryos. Plus IP from Kc167 cells.

Quote of the Week: Spring is for Gardening

Have you a pond in your garden? If not, why not think about making one? ... If you do this you won't have to spend much time gardening, and you will have so much more time to watch insects! ... You will not need to find insects to put in your pond; they will come by themselves.

From The Insects in Your Garden, Harold Oldroyd, 1976, Kestrel Books

Breaking Report--DRSC Screen Data Published

What's on my desk to read today?  

Sessions et al. (2009) Discovery of insect and human dengue virus host factors. Nature 458:1047-1050. And see Editor's Note in the same issue of the journal.

This screen was performed at the DRSC using our whole-genome dsRNA library.

In the paper, the authors report the results of the Drosophila screen. Plus follow-up in human cells--they used siRNAs to target homologs of the fly genes they identified--and in the dengue virus vector mosquito Ae. aegypti--which they inject with dsRNA. The authors report "notable conservation of required factors between dipteran [fly & mosquito] and human hosts."

Using RNAi in three species to get closer to combating an infectious disease endemic in three continents and for which there is currently no vaccine or cure? Pretty darned cool.

Rumor Has It ...

A new section in Genetics? I'm anxiously awaiting the May issue for an announcement.

Breaking Report: single-cell RNA-Seq

On my desk today? Tang et al. (2009) mRNA-Seq whole-transcriptome analysis of a

single cell in Nature Methods.

Why this paper? At the protocol level, it's good to see what's resulting from availability of next generation sequencing technologies. And at the biological level, interesting to note that the authors found that about 1500 genes were up-regulated in Dicer1 -/- or Ago2 -/- mouse oocytes (with about 600 up-regulated in both genetic backgrounds).

Breaking Report: VDRC Collection full-genome RNAi Screen

Reading today? Mummery-Widmer et al. (2009) Genome-wide analysis of Notch signalling in Drosophila by transgenic RNAi. Nature. PubMed ID 19363474.

Protocol notes:  Genome-wide screen with transgenic RNAi lines from the Vienna Drosophila RNAi Center (see VDRC). More detailed information about screening and follow-up tests can be found in the footnotes to the supplementary tables.

Breaking Reports: RNAi Screens in Mammalian ES Cells

What's on my desk today?  Reports of genome-scale RNAi screens in mammalian embryonic stem cells:

Ding et al. (2009) A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity. Cell Stem Cell.

Hu et al. (2009) A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal. Genes & Development (free full text).

Why these two papers? They are among the best-controlled large-scale mammalian RNAi screens I've seen.  In Ding et al. they use esiRNAs to screen and follow up with verification of gene expression by RT-PCR; test with independent esiRNAs against primary hits; tests in another cell line; additional cell-based assays; cross-species rescue; and integration of ChIP & mass spec data. Similarly, in Hu et al. they use pooled siRNAs to screen and follow up with test of individual siRNAs; expression analysis via Affymetrix chips; qPCR to verify knockdown; additional cell-based assays; and integration of ChIP-chip data.

Quote of the Week: Talking Flies

Flies are among the most familiar of insects, and yet among the least understood. We talk about 'the fly', as we talk about 'the ant' and 'the frog', and then we have in mind one species, the common house-fly, Musca domestica. ... We notice other flies when they are a nuisance to us. Bluebottles and other blow-flies taint our meat; flies swarm round our heads and those of our grazing animals in summer; some bite, some buzz, some just disgust us with their habit of breeding in dung, sewage, carrion, even in living flesh. In fact, flies are a topic like drains, not to be discussed in polite society, to be left to those strange people who cultivate a professional interest in them. It is a pity.

Harold Oldroyd (1964), The Natural History of Flies

Breaking Report: Conditional Mutagenesis

What am I reading today?  A brevia report in Science from the Hassan lab.  Choi et al. (2009) Conditional Mutagenesis in Drosophila. Science 324: 54. PubMed ID 19342580. The authors report their "integrase-mediated approch for gene knock-out."

A Look at the DRSC: Cell Culture

The cell culture facility at the DRSC is modest in size (this wide-angle photo is generous to the space).  But it's also more than sufficient. 

We have two sterile hoods, one of which is set up to make things easier for a left-handed staff member. We also have an incubator, a Leica 'scope, a fridge, and a few other necessities. 

Culturing fly cells is similar to culturing mammalian cells. Fly cells have an advantage in that they don't require the same carbon dioxide-rich environment you have to pipe in for mammalian cells. Humidity and 25 degrees C does the trick.

TRiP at Bloomington

Rumor has it the Transgenic RNAi Project (TRiP) "toolbox" stocks are a popular request at the Bloomington stock center now that they are available there (along with an increasing number of TRiP stocks with hairpins directed against specific genes).

Quote of the Week: Cell Line Selection

I was asked this week about choosing a cell line appropriate to a specific assay. 

One thing to think about is the biology of the cells. Ask yourself, Does the cell line have the characteristics necessary for the topic I want to address? Express the right genes (or can I add them)? Respond to the stimulus? 

But practical concerns are also important. Although S2 is a popular choice for screens at the DRSC, the choice for an imaging screen is often S2R+, as these cells tend to adhere better to the bottom of the plate, an important feature for automated imaging at 384-well format.

Today's quote is from Section 1.1., Identifying a Suitable Cell Line, in B. Baum & L. Cherbas (2007) Drosophila Cell Lines as Model Systems and as an Experimental Tool.  In Drosophila: Methods & Protocols. C. Dahmann, Ed. Methods in Molecular Biology series Vol. 420. ISBN: 978-1-58829-817-1.

More than 100 cell lines have been established from Drosophila embryos and larvae over the last 40 yr. The first of these, including the commonly used S2 and Kc lines (10–12), were generated from spontaneously immortalized cells in cultures of mechanically dissociated embryos. In subsequent years, similar methods were used to derive cell lines from the early embryos of genetically defined fly stocks (11,13–15). Interestingly, these embryonic lines all share characteristics that suggest that they are derived from immortalized hematopoietic cells. This idea is supported by the similarities of these lines to mbn-2 and mbn 3 lines that were established from primary cultures of mutant embryos carrying blood cell tumors (16). Furthermore, the S2R+ cell line, an isolate of the S2 cell line (17), bears a striking resemblance in form and behavior to larval hemocytes isolated by bleeding larvae.

Please see the full chapter for reference citations.

Over-Stimulated

What am I reading this week? Not much more than requests for applications (RFAs) and flurries of emails related to the stimulus funds at NIH. When things calm down I hope to have something more scientifically relevant to say here. To my fellow U.S. scientists (and anyone else in the throws of grant-writing), best of luck and many happy revisions to text.

Relevant link: NIH and the American Recovery & Reinvestment Act

Quote of the Week: If only ...

I recently overheard a now familiar conversation, one fly biologist lamenting to another that there is no feasible way to cryopreserve flies the way one can do with C. elegans, mammalian cells and (thankfully for the DRSC) cultured fly cell lines. But perhaps--given what some other flies can do--there is still hope for some solution to endless stock flipping?

... the larvae of Polypedilum vanderplanki can be completely dehydrated, and will enter into a state of 'cryptobiosis' during which all metabolic processes apparently cease ... The larva consumes no oxygen--it will survive in an atmosphere of pure nitrogen and it can withstand injuries that in normal life would set up a chain of disturbances that would kill the larva. When it is put back into water it quickly swells to normal size, and quietly resumes its breathing and feeding.

H. Oldryod, The Natural History of Flies

Breaking Report & on Small Libraries

On my desk today? A report from Ardehali et al. (2009) Spt6 enhances the elongation rate of RNA polymerase II in vivo. EMBO Journal.

Protocol Notes: They screened a set of 141 candidate transcription factors in S2 cells. Not clear to me what dsRNAs they used (their own? is in supplement?). Screen follow-up using a transgenic RNAi fly line (670-bp Spt6 fragment amplified and cloned "head-to-head" into pWIZ).

Makes me think about: A couple of things available at the DRSC. 

First, we have a Transcription Factor sub-library set that contains more than 900 putative transcription factors. Why so many? The list is generous with its definition of a TF. 

If you catch us before we have the downloadable list of genes in the set up on the website, no problem, drop me an email and I'll send the file--we openly share the gene lists.

Second, although you might be interested to screen a small library of candidates, we recognize that making those dsRNAs may be new to you. And a hurdle you're not anxious to jump. To help out we are now offering a custom dsRNA small library production service. 

Send us your gene list (one or a few 96-well plates worth) or a list of our amplicon IDs. We'll cherry-pick the corresponding PCR amplicons in our collection. And use those as template to make up a fresh batch of dsRNA in whatever plate format you'd like. We'll provide enough dsRNA for several assays. 

We announced the custom dsRNA service at the fly conference and have done two pilot projects (3 96-well plates each). But it's not up on our website as of this posting. Drop me an email if you're interested to know the fee or have questions.

Transfection

The DRSC Protocols Pages describe our current methods for things like cell culture, transfection, dsRNA production and setting up a 384-well format screen. 

Today I'm thinking most about transfection. As with any protocol, I'm interested in two things. How to do it well. And how to do it well cheaply--particularly important when you're doing things at high-throughput scale as researchers do here for whole-genome screens.

Based on experience, we've been using Qiagen Effectene as a transfection reagent.  Some visiting screeners use a reagent from Invitrogen. And I recently got a protocol from the folks at Genesee Scientific for FectoFly (made in Europe by PolyPlus).

Have feedback? Experience? Favorites? We are interested to hear about it. So we can provide the best advice on the DRSC website and to visiting researchers.

RNAi in Whole Animals

The DRSC was originally focused on screening in cells. And we keep going down that path.

But we're moving in a new direction as well. Thanks to the Transgenic RNAi Project (TRiP). 

Shown here, the TRiP fly-pushing stations, which are being used for stock maintenance, making homozygous transgenic lines and more. 

Visit the TRiP site to view a list of available lines. 

More and more of these will be available at Bloomington soon (some are there already). In the meantime they can be requested from the TRiP directly. You can also read on-line about the TRiP strategy and plans, community nominations for genes TRiP should target, and screening the TRiP lines at the DRSC. 

Related links:

Bloomington Drosophila Stock Center

Vienna Drosophila RNAi Center

Quote of the Week: Philosophizing

In an uncertain environment, good intuitions must ignore information.

Gerd Gigerenzer in Gut Feelings: The Intelligence of the Unconscious (2007, Penguin Group USA)

Do cells do this (i.e. focus in on key pieces of information at the expense of other information prior to making decisions)? Do scientists? Should they?

Things to come ...

At the fly meeting, I stopped by the FlyBase demo room.  Was happy to learn that some time this year, FlyBase will be launching information pages for commonly used cell lines (like S2) and their derivatives. Will post here when I've learned that the pages have launched. 

Between this and cell line expression data being generated by the modENCODE project, there should soon be a lot more help for folks looking to make informed choices about what cell line to choose for a particular cell screening project. Such as by learning in what cells key components of a pathway of interest are expressed.

Breaking Report--Cell vs. Cell vs. Cell

On my desk today? A paper reporting the results of doing the same screen in several Drosophila cell types.

Liu, Sims & Baum (2009) A parallel RNAi screen across different cell lines identifies generic and cell-type specific regulators of actin organisation and cell morphology. Genome Biology 10:R26.

Protocol Notes: They designed and built a kinome RNAi library based on gene-specific primers in their FLIGHT db or using the DFKZ E-RNAi tool. Kc167, S2R+, S2, BG2-c2, BG3-c2 and BG3-c1 lines tested. Microtubule, actin and DNA staining read-outs with imaging.

Reminds me of:  Some papers that describe the results of cross cell-type screens in mammalian cells. I am aware of at least two such reports (please comment if you know of others).

Grueneberg et al. (2008) Kinase requirements in human cells: I. Comparing kinase requirements across various cell types. PNAS 105(43):16472-7.

Luo et al. (2008) Highly parallel identification of essential genes in cancer cells. 105(51):20380-5.

Tango Time

What I'm reading today? Well, not much! As I'm working to catch up after a late night flight back from the fly meeting. I am trying to make time for this one:

Saito et al. (2009) TANGO1 facilitates cargo loading at edoplasmic reticulum exit sites. Cell 136:891-902. Of technical note, includes collagen secretion and metabolic assay read-outs after siRNA treatment of mammalian cells.

It's a lovely follow-up on a paper that describes screen data obtained at the DRSC, Bard et al. (2006) Functional genomics reveals genes involved in protein secretion and Golgi organization. Nature 439:604-607.

If you were at the meeting, hope that like me you enjoyed it and learned much. Abstracts available as downloadable PDFs at the ADRC website (see "Complete Volume" and "Program Adendum").

50th ADRC

If you're at the fly conference, stop by and say hello!  Posters 864C & 868A.

More Aspiration, Less Perspiration

We're excited for our latest "toy"  (pictured here).  

Why do we care? There are more than 60 plates in the DRSC 2.0 full-genome library, which is the library in use for all full-genome screens at the DRSC (optimized to limit off-target effects, covers about 14,000 genes, includes protein-coding and non-coding genes). 

Add to that, screens are done in duplicate, and suddenly you're looking at adding and aspirating small volumes to more than 100 384-well plates for a full-genome screen. 

As a result, it's great to find automated and all-at-once ways to do repetitive steps like liquid addition (for which we have several automated instruments) and aspiration.  Making it possible to process more plates per day, such that a screen takes weeks instead of months.

What we used to use?  An aspirator "wand" that can be used to aspirate fluid from a 384-well plate a row at a time. Two minuses: (1) only a row at a time! And (2) you had to hold it very steady and judge by eye and feel if you hit the right depth each time.

What's better about this? With the new device, you pre-set the height you'll bring the plate up to. This means you can adjust the device for any plate type and any pin depth (good). Once it's adjusted, you get the same depth in each well and plate as you go (great!).  And of course, because there are 384 pins, a whole plate is done in one quick go.

Cell Special Issue on RNA

Of course using RNAi screening to study some topic and the study of RNAi (or RNA more generally) are not one and the same.  

But we put ourselves in the best position for screening--in terms of reagent development and data interpretation, for example--by keeping up on at least some of the wealth of info that's coming out about RNA.

With this in mind, what's on my desk today?  The special issue on RNA from Cell.  Cover photo shown here.

Quote of the Week: Comparing Screen and Array Datasets

Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens. By comparing the results of these two assays [in yeast] across various stimuli, we found that genetic screens tend to identify response regulators, whereas mRNA profiling frequently detects metabolic responses.

Yeger-Lotem et al. (2009) Bridging high-throughput genetic and transcriptional data reveals cellular responses to alpha-synuclein toxicity. Nature Genetics. 41(3):316-323. PubMed ID: 19234470.

A Look at the DRSC: Main Lab

Breaking Paper--Control of Cell Size

On my desk today? Sims et al. (2009) PDGF/VEGF signalling controls cell size in Drosophila. Genome Biology. PubMed ID 19216764.  As of this posting, abstract viewable on-line and free full text downloadable as provisional PDF.

The paper focuses on cell size-related results from a genome-wide screen performed at the DRSC (see also Boutros et al. 2004 Science 303:832-835). 

Protocol Notes:  Screen done with S2R+ cells. Image data were put through an automated image analysis to determine average cell size (total monolayer cell area divided by the number of DAPI-positive nuclei counts). With by-eye follow up to check for phenotypes like cell adhesion effects that were not of interest for this study but might also score as positives. Network analysis using FLIGHT. Hit validation using two independent amplicons and a related but different image-based analysis.

Quote of the Week--Act III, Scene 2

Poor harmless fly,

That with his pretty buzzing melody

Came here to make us merry!

William Shakespeare, Titus Andronicus

Web Wednesday--50th ADRC Abstracts On-Line

Today's web feature is the Abstract Planner, now on-line in advance of the upcoming 50th Annual Drosophila Research Conference in Chicago, IL, USA.  Search away!  And remember, in an effort to go green, this year's conference booklets will not have full abstracts.

Breaking Report--Ecdysone & Death

On my desktop today?  

A report from Chittaranjan et al. (2009) Steroid Hormone Control of Cell Death and Cell Survival: Molecular Insights Using RNAi. Free online access from PLoS Genetics. (Figure show is from this report.)

Like the report from Rogers et al. that I blogged about in a previous post (Little Screens, Big Results), these authors focused their screen on a set of candidate genes. In this case, a set of 460 candidates. To do this, the authors generated dsRNAs for each candidate and followed up with a second dsRNA for screen hits.

Chitteranjan et al. performed what is essentially two screens. One focused on identification of pro-death and pro-surivival factors. And the other on "death related effects" dependent upon or independent of ecdysone treatment.

Protocol notes:  Their screen was done on ecdysone-treated l(2)mbn cells. Death and survival monitored with what I think of as an old-school (but reliable) approach, a trypan blue exclusion assay. And they took a closer look at cell death with DAPI staining and TUNEL analysis.

Some thoughts:  Reading another report like this focused on a defined set of candidate genes has me excited for something the DRSC has just begun to offer:  synthesis of custom sets of dsRNAs in 96-well format (based on existing amplicons).  

This made sense as (1) we're expert at doing IVTs at high-throughput, including quality analysis and troubleshooting, and have ready access to our collections of PCR amplicons used as templates for the dsRNA. And (2) the increasing interest in the community to screen a custom-curated sets of genes.  

We just successfully completed a pilot project and given the right lead-time, can now accommodate more requests. 

Please contact me if you're interested to learn more about the service.