RNAi screening, double RNAi and multi parametric image assays used to build map of cell cycle regulators

Billmann M, Horn T, Fischer B, Sandmann T, Huber W, Boutros M. A genetic interaction map of cell cycle regulators. Mol Biol Cell. 2016 Feb 24. PMID: 26912791.

From the abstract: "Cell based RNAi is a powerful approach to screen for modulators of many cellular processes. However, resulting candidate gene lists from cell-based assays comprise diverse effectors, both direct and indirect, and further dissecting their functions can be challenging. Here, we screened a genome-wide RNAi library for modulators of mitosis and cytokinesis in Drosophila S2 cells. ... We then characterized ∼300 candidate modifiers further by genetic interaction analysis using double RNAi and a multiparametric, imaging-based assay. ... Our results show that the combination of genome-scale RNAi screening and genetic interaction analysis using process-directed phenotypes provides a powerful two-step approach to assign components to specific pathways and complexes."

S2 cells used to express human HER2 protein domains

Kanthala S, Mill CP, Riese DJ 2nd, Jaiswal M, Jois S. Expression And Purification Of HER2 Extracellular Domain Proteins In Schneider2 Insect Cells. Protein Expr Purif. 2015 Sep 9. PMID: 26363121.

RNAi and RNA analysis. Cell cycle study. Breaking report.

These authors follow RNAi screening for DNA content in S2 cells with transcriptional profiling to uncover transcriptional networks. Bonke M, Turunen M, Sokolova M, Vähärautio A, Kivioja T, Taipale M, Björklund M, Taipale J. Transcriptional networks controlling the cell cycle. G3 (Bethesda). 2013 Jan;3(1):75-90. doi: 10.1534/g3.112.004283. PubMed PMID: 23316440.

Breaking Report: in vivo RNAi Screen Focused on Chromosome Instability

On my desk to read today? This open access report on an in vivo RNAi screen.

Shaukat Z, Wong HW, Nicolson S, Saint RB, Gregory SL. A screen for selective killing of cells with chromosomal instability induced by a spindle checkpoint defect. PLoS One. 2012;7(10):e47447. doi: 10.1371/journal.pone.0047447. PMID: 23077619.

Breaking Report: Moesin Dephosphorylation

On my desk to read today? A report of an RNAi screen focused on phosphatases.

Kunda et al. 2012 PP1-Mediated Moesin Dephosphorylation Couples Polar Relaxation to Mitotic Exit. Curr Biol. ePubed 2011 Dec 28. PubMed ID: 22209527

Methods Papers

There are at least two papers in volume 782 of Methods in Molecular Biology that may be relevant to cell-based Drosophila RNAi screening:

Davidson JM, Duronio RJ. (2011) Using Drosophila S2 Cells to Measure S phase-Coupled Protein Destruction via Flow Cytometry. Methods Mol Biol. 782:205-219. PMID: 21870294.

Siudeja K, de Jong J, Sibon OC. (2011) Studying Cell Cycle Checkpoints Using Drosophila Cultured Cells. Methods Mol Biol. 782:59-73. PMID: 21870285.

Cross-Species Validation with Live Imaging--Cell Cycle Screen Hits

Also on my desk today? A report from Sironi et al. using living mammalian cell imaging and analysis to follow up on hits from RNAi screens related to the cell cycle performed in other species.

Sironi et al. (2011) Automatic quantification of microtubule dynamics enables RNAi-screening of new mitotic spindle regulators. Cytoskeleton 68:266-278.

Breaking Report: Power of Combinatorial RNAi

On my desk to read today? A new report from T. Horn and colleagues in the Boutros group on the use of double-knockdown to uncover genetic interactions.

Horn et al. (2011) Mapping of signaling networks through synthetic genetic interaction analysis by RNAi. Nature Methods. Advanced online publication. PMID: 21378980.

More than 70,000 measurements!

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.

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.

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.

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).

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.