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).