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.