Friday, April 22, 2016

Combining high-throughput RNAi with RNA expression proves powerful

Zanotto-Filho A, Dashnamoorthy R, Loranc E, de Souza LH, Moreira JC, Suresh U, Chen Y, Bishop AJ. Combined Gene Expression and RNAi Screening to Identify Alkylation Damage Survival Pathways from Fly to Human. PLoS One. 2016 Apr 21;11(4):e0153970. PMID: 27100653.

From the abstract: "... We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray) prior to analysis... From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival."

Tuesday, April 19, 2016

VDRC announces additional 500 RNAi fly stocks available

The VDRSC has announced that additional new RNAi fly stocks using shRNA technology like that used by the Trangenic RNAi Project (TRiP) are now available. In their news item of April 18, 2016, they state that 200 new lines are available and 300 more will be added.

For details see VDRC's news announcement on their website.

Friday, April 15, 2016

High-throughput assay for individual fly egg laying counts reported

Gou B, Zhu E, He R, Stern U, Yang CH. High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster. J Vis Exp. 2016 Mar 24;(109). PMID: 27077482.

From the abstract: "Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ... To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate ... This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers."

Friday, March 18, 2016

Persistence of RNAi in mosaic studies

Another report regarding in vivo RNAi that fly folks using the technique should be aware of (see this post for another, and check out other posts tagged "in vivo RNAi" or "troubleshooting").

Bosch JA, Sumabat TM, Hariharan IK. Persistence of RNAi-Mediated Knockdown in Drosophila Complicates Mosaic Analysis yet Enables Highly Sensitive Lineage Tracing. Genetics. 2016 Mar 16. PMID: 26984059.

From the abstract: "... By expressing synthetic short hairpin RNAs (shRNAs) using the Gal4/UAS system, knockdown is efficiently achieved in specific tissues or in clones of marked cells. ... knockdown by shRNAs is so potent and persistent that even transient exposure of cells to shRNAs can reduce gene function in their descendants. When using the FLP-out Gal4 method, in some instances we observed unmarked "shadow RNAi" clones adjacent to Gal4-expressing clones, which may have resulted from brief Gal4 expression following recombination but prior to cell division. Similarly, Gal4 driver lines with dynamic expression patterns can generate shadow RNAi cells after their activity has ceased ... Importantly, these effects can lead to erroneous conclusions regarding the cell autonomy of knockdown phenotypes. We have investigated the basis of this phenomenon and suggested experimental designs for eliminating ambiguities in interpretation. We have also exploited the persistence of shRNA-mediated knockdown to design a sensitive lineage-tracing method, i-TRACE, which is capable of detecting even low levels of past reporter expression. ..."

Sunday, February 28, 2016

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."

RNA pol II Cdk12 identified as an Nrf2 target via RNAi screening in S2 cells

Li X, Chatterjee N, Spirohn K, Boutros M, Bohmann D. Cdk12 Is A Gene-Selective RNA Polymerase II Kinase That Regulates a Subset of the Transcriptome, Including Nrf2 Target Genes. Sci Rep. 2016 Feb 25;6:21455. PMID: 26911346.

From the abstract: "The Nrf2 transcription factor is well conserved throughout metazoan evolution and serves as a central regulator of adaptive cellular responses to oxidative stress. We carried out an RNAi screen in Drosophila S2 cells to better understand the regulatory mechanisms governing Nrf2 target gene expression. This paper describes the identification and characterization of the RNA polymerase II (Pol II) kinase Cdk12 as a factor that is required for Nrf2 target gene expression in cell culture and in vivo. ... We suggest that Cdk12 acts as a gene-selective Pol II kinase that engages a global shift in gene expression to switch cells from a metabolically active state to "stress-defence mode" when challenged by external stress."