Friday, December 9, 2016

From the Department of Very Specific Phenotypes: in vivo RNAi screen related to formation of protrusions on the corneal lens of the fly eye

Minami R, Sato C, Yamahama Y, Kubo H, Hariyama T, Kimura KI. An RNAi Screen for Genes Involved in Nanoscale Protrusion Formation on Corneal Lens in Drosophila melanogaster. Zoolog Sci. 2016 Dec;33(6):583-591. PMID: 27927092.

From the abstract: "The "moth-eye" structure, which is observed on the surface of corneal lens in several insects, supports anti-reflective and self-cleaning functions due to nanoscale protrusions ... the mechanism of protrusion formation from cell-secreted substances is unknown. ... In the present study, we sought to identify genes involved in "moth-eye" structure, formation in order to elucidate the developmental mechanism of the protrusions in Drosophila. We re-examined the aberrant patterns in classical glossy-eye mutants by scanning electron microscope and classified the aberrant patterns into groups. Next, we screened genes encoding putative structural cuticular proteins and genes involved in cuticular formation using eye specific RNAi silencing methods combined with the Gal4/UAS expression system. We identified 12 of 100 candidate genes, such as cuticular proteins family genes (Cuticular protein 23B and Cuticular protein 49Ah), cuticle secretion-related genes (Syntaxin 1A and Sec61 ββ subunit), ecdysone signaling and biosynthesis-related genes (Ecdysone receptor, Blimp-1, and shroud), and genes involved in cell polarity/cell architecture (Actin 5C, shotgun, armadillo, discs large1, and coracle). Although some of the genes we identified may affect corneal protrusion formation indirectly through general patterning defects in eye formation, these initial findings have encouraged us to more systematically explore the precise mechanisms underlying the formation of nanoscale protrusions in Drosophila."

Tuesday, November 1, 2016

Methods reviews for in vivo RNAi screening in Drosophila

Four! RNAi-relevant methods papers in the recent Drosophila-focused issue of Methods in Molecular Biology:

Billmann M, Boutros M. Methods for High-Throughput RNAi Screening in Drosophila Cells. Methods Mol Biol. 2016;1478:95-116. PMID: 27730577.

Kaya-Çopur A, Schnorrer F. A Guide to Genome-Wide In Vivo RNAi Applications in Drosophila. Methods Mol Biol. 2016;1478:117-143. PMID: 27730578.

Zhou J, Tong C. Design and Methods of Large-Scale RNA Interference Screens in Drosophila. Methods Mol Biol. 2016;1470:163-9. PMID: 27581292. Chen X, Xu L.

Genome-Wide RNAi Screening to Dissect the TGF-β Signal Transduction Pathway. Methods Mol Biol. 2016;1344:365-77. PMID: 26520138.

Monday, October 31, 2016

RNAi used to induce ROS stress in a study of oogenesis

Perkins AT, Das TM, Panzera LC, Bickel SE. Oxidative stress in oocytes during midprophase induces premature loss of cohesion and chromosome segregation errors. Proc Natl Acad Sci U S A. 2016 Oct 17. pii: 201612047. PMID: 27791141.

From the abstract: "... A growing body of evidence suggests that meiotic cohesion deteriorates as oocytes age ... One hallmark of aging cells is an increase in oxidative damage caused by reactive oxygen species (ROS). Therefore, increased oxidative damage in older oocytes may be one of the factors that leads to premature loss of cohesion and segregation errors. To test this hypothesis, we used an RNAi strategy to induce oxidative stress in Drosophila oocytes and measured the fidelity of chromosome segregation during meiosis. ..."

Tick tock! Screening the clock.

Agrawal P, Hardin PE. An RNAi Screen to Identify Protein Phosphatases that Function Within the Drosophila Circadian Clock. G3 (Bethesda). 2016 Oct 26. PMID: 27784754.

From the abstract: "Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. ... To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. ... Additional RNAi lines, transposon inserts, overexpression and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. ... 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans."

Thursday, October 13, 2016

Methods publications relevant to cell and in vivo RNAi

Methods in Molecular Biology has recently published papers relevant to Drosophila cell culture, cell-based RNAi, and in vivo RNAi.

Debec A, Megraw TL, Guichet A. Methods to Establish Drosophila Cell Lines. Methods Mol Biol. 2016;1478:333-351. PubMed PMID: 27730593

Billmann M, Boutros M. Methods for High-Throughput RNAi Screening in Drosophila Cells. Methods Mol Biol. 2016;1478:95-116. PubMed PMID: 27730577.

Kaya-Çopur A, Schnorrer F. A Guide to Genome-Wide In Vivo RNAi Applications in Drosophila. Methods Mol Biol. 2016;1478:117-143. PubMed PMID: 27730578.

Monday, September 12, 2016

Drosophila CRISPR protocols series

Thinking fly CRISPR? Series of methods papers out in CSH Protocols.

Housden BE, Perrimon N. Cas9-Mediated Genome Engineering in Drosophila melanogaster. Cold Spring Harb Protoc. 2016 Sep 1;2016(9):pdb.top086843. PMID: 27587786.

Housden BE, Perrimon N. Detection of Indel Mutations in Drosophila by High-Resolution Melt Analysis (HRMA). Cold Spring Harb Protoc. 2016 Sep 1;2016(9):pdb.prot090795. PMID: 27587781.

Housden BE, Perrimon N. Design and Generation of Donor Constructs for Genome Engineering in Drosophila. Cold Spring Harb Protoc. 2016 Sep 1;2016(9):pdb.prot090787. PMID: 27587780.

Housden BE, Hu Y, Perrimon N. Design and Generation of Drosophila Single Guide RNA Expression Constructs. Cold Spring Harb Protoc. 2016 Sep 1;2016(9):pdb.prot090779. PMID: 27587779.

Tuesday, September 6, 2016

Housden BE, Hu Y, Perrimon N. Design and Generation of Drosophila Single Guide RNA Expression Constructs. Cold Spring Harb Protoc. 2016 Sep 1;2016(9):pdb.prot090779. PMID: 27587779.

From the abstract: "... Here, we provide a step-by-step protocol for designing sgRNA target sites using the Drosophila RNAi Screening Center (DRSC) Find CRISPRs tool (version 2). We also describe the generation of sgRNA expression plasmids for the use in cultured Drosophila cells or in vivo. Finally, we discuss specific design requirements for various genome engineering applications."

Methods paper -- in vivo RNAi screens

Zhou J, Tong C. Design and Methods of Large-Scale RNA Interference Screens in Drosophila. Methods Mol Biol. 2016;1470:163-9. PMID: 27581292.

From the abstract: "... Here, we discuss methods for the design and performance of a large-scale in vivo RNAi screen in Drosophila. Furthermore, methods for the validation of results and analysis of data will be introduced."

Monday, August 22, 2016

Using the white gene as an indicator of CRISPR-based editing

Ge DT, Tipping C, Brodsky MH, Zamore PD. Rapid Screening for CRISPR-Directed Editing of the Drosophila Genome Using white Co-Conversion. G3 (Bethesda). 2016 Aug 19. pii: g3.116.032557. PMID: 27543296.

From the abstract: "... Here, we describe a strategy that reduces the time and effort required to identify flies with targeted genomic changes. The strategy uses editing of the white gene, evidenced by altered eye color, to predict successful editing of an unrelated gene-of-interest. The red eyes of wild-type flies are readily distinguished from white-eyed (end joining-mediated loss of White function) or brown-eyed (recombination-mediated conversion to the whitecoffee allele) mutant flies. ... We find that end joining-mediated mutations often show signatures of microhomology-mediated repair and that recombination-based mutations frequently involve donor plasmid integration at the target locus. Finally, we show that gap repair induced by two guide RNAs more reliably converts the intervening target sequence, whereas the use of Lig4169 mutants to suppress end joining does not improve recombination efficacy."

Genome-wide screen in study of heterochromatin factor HP1a

The cell-based RNAi screen described in this study was done using a DRSC library and follow-up reagents. Congratulations, Joel and colleagues!

Swenson JM, Colmenares SU, Strom AR, Costes SV, Karpen GH. The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic. Elife. 2016 Aug 11;5. pii: e16096. PMID: 27514026; PMCID: PMC4981497.

Abstract: "Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions."

Friday, August 5, 2016

Genome-wide in vivo screen looks at male germline stem cells

Liu Y, Ge Q, Chan B, Liu H, Singh SR, Manley J, Lee J, Weideman AM, Hou G, Hou SX. Whole-animal genome-wide RNAi screen identifies networks regulating male germline stem cells in Drosophila. Nat Commun. 2016 Aug 3;7:12149. PMID: 27484291.

From the abstract: "... To identify genes and pathways that regulate stem-cell fates in the whole organism, we perform a genome-wide transgenic RNAi screen through ubiquitous gene knockdowns, focusing on regulators of adult Drosophila testis germline stem cells (GSCs). Here we identify 530 genes that regulate GSC maintenance and differentiation. Of these, we further knock down 113 selected genes using cell-type-specific Gal4s and find that more than half were external regulators, that is, from the local microenvironment or more distal sources. ..."

Talk of transvection, RNAi, and Gal4-UAS, all in one G3 report

Tuesday, July 5, 2016

DRSC & TRiP website -- All new!

We just went live with a completely redone DRSC & TRiP website.

And a new name, DRSC/TRiP Functional Genomics Resources.

The pages will re-direct (except for online tools, which will still live at that URL).

Or you can check it out directly at

Feedback welcome!

Thursday, June 30, 2016

Gene editing in fly cells--new report from Kunzelmann et al. in G3

Kunzelmann S, Böttcher R, Schmidts I, Förstemann K. A Comprehensive Toolbox for Genome Editing in Cultured Drosophila melanogaster Cells. G3 (Bethesda). 2016 Jun 1;6(6):1777-85. PMID: 27172193

From the abstract: "... Following up on our initial publication, we now describe a considerably simplified, more efficient, and readily scalable experimental workflow for PCR-based genome editing in cultured Drosophila melanogaster cells. Our analysis at the act5C locus suggests that PCR-based homology arms of 60 bp are sufficient to reach targeting efficiencies of up to 80% after selection; extension to 80 bp (PCR) or 500 bp (targeting vector) did not further improve the yield. We have expanded our targeting system to N-terminal epitope tags; this also allows the generation of cell populations with heterologous expression control of the tagged locus via the copper-inducible mtnDE promoter. We present detailed, quantitative data on editing efficiencies for several genomic loci that may serve as positive controls or benchmarks in other laboratories. ..."

EP-MS2 method and RNAi follow-up used in screen for localized RNAs

Misra M, Edmund H, Ennis D, Schlueter MA, Marot JE, Tambasco J, Barlow I, Sigurbjornsdottir S, Mathew R, Vallés AM, Davis I, Leptin M, Gavis ER. A Genome-Wide Screen for Dendritically Localized RNAs Identifies Genes Required for Dendrite Morphogenesis. G3 (Bethesda). 2016 Jun 3. pii: g3.116.030353. PMID: 27260999.

From the abstract: "Localizing messenger RNAs at specific subcellular sites is a conserved mechanism for targeting the synthesis of cytoplasmic proteins to distinct subcellular domains ... However, the full range of transcripts that are asymmetrically distributed in specialized cell types and the significance of their localization, especially in the nervous system, are not known. We used the EP-MS2 method, which combines EP transposon insertion with the MS2/MCP in vivo fluorescent labeling system to screen for novel localized transcripts in polarized cells, focusing on the highly branched Drosophila class IV dendritic arborization neurons. Of a total of 541 lines screened, we identified 55 EP-MS2 insertions producing transcripts that were enriched in neuronal processes, particularly in dendrites. The 47 genes identified by these insertions encode molecularly diverse proteins and are enriched for genes that function in neuronal development and physiology. RNAi-mediated knockdown confirmed roles for many of the candidate genes in dendrite morphogenesis. ..."

Tuesday, June 28, 2016

in vivo RNAi screen related to cholesterol and steroid hormones

Danielsen ET, Moeller ME, Yamanaka N, Ou Q, Laursen JM, Soenderholm C, Zhuo R, Phelps B, Tang K, Zeng J, Kondo S, Nielsen CH, Harvald EB, Faergeman NJ, Haley MJ, O'Connor KA, King-Jones K, O'Connor MB, Rewitz KF. A Drosophila Genome-Wide Screen Identifies Regulators of Steroid Hormone Production and Developmental Timing. Dev Cell. 2016 Jun 20;37(6):558-70. PMID: 27326933; PMCID: PMC4918455.

From the abstract: "Steroid hormones control important developmental processes and are linked to many diseases. To systematically identify genes and pathways required for steroid production, we performed a Drosophila genome-wide in vivo RNAi screen and identified 1,906 genes with potential roles in steroidogenesis and developmental timing. Here, we use our screen as a resource to identify mechanisms regulating intracellular levels of cholesterol, a substrate for steroidogenesis. ... In addition, we demonstrate the existence of an autophagosomal cholesterol mobilization mechanism and show that activation of this system rescues Niemann-Pick type C1 deficiency that causes a disorder characterized by cholesterol accumulation. ... These results reveal genes regulating steroidogenesis during development that likely modulate disease mechanisms."

Friday, June 10, 2016

Combined CRISPR-RNAi approach

On my desk to read today? This report from former Perrimonian and DRSC screener R. Neumuller:

Wissel S, Kieser A, Yasugi T, Duchek P, Roitinger E, Gokcezade JF, Steinmann V, Gaul U, Mechtler K, Förstemann K, Knoblich JA, Neumüller RA. A Combination of CRISPR/Cas9 and Standardized RNAi as a Versatile Platform for the Characterization of Gene Function. G3 (Bethesda). 2016 Jun 8. pii: g3.116.028571. PMID: 27280787.

From the abstract: "... miGFPi combines CRISPR/Cas9 mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method ... In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. ..."

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."

Monday, February 15, 2016

Genome-wide cell-based screen related to microtubule bundling and lysosome motility

Jolly AL, Luan CH, Dusel BE, Dunne SF, Winding M, Dixit VJ, Robins C, Saluk JL, Logan DJ, Carpenter AE, Sharma M, Dean D, Cohen AR, Gelfand VI. A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells. Cell Rep. 2016 Jan 26;14(3):611-20. PMID: 26774481.

Cell-based RNAi screen looks at TGF-beta signaling--methods-focused report

Chen X, Xu L. Genome-Wide RNAi Screening to Dissect the TGF-β Signal Transduction Pathway. Methods Mol Biol. 2016;1344:365-77. PMID: 26520138.

From the abstract: "... Here, we describe a protocol for image-based whole-genome RNAi screening aimed at identifying molecules required for TGF-β signaling into the nucleus. Using this protocol we examined 90% of annotated Drosophila open reading frames (ORF) individually and successfully uncovered several novel factors serving critical roles in the TGF-β pathway. Thus cell-based high-throughput functional genomics can uncover new mechanistic insights on signaling pathways beyond what the classical genetics had revealed."

Tuesday, February 9, 2016

Analysis of steroid hormone signaling in 41 Drosophila fly cell lines

Stoiber M, Celniker S, Cherbas L, Brown B, Cherbas P. Diverse Hormone Response Networks in 41 Independent Drosophila Cell Lines. G3 (Bethesda). 2016 Jan 15. pii: g3.115.023366. PMID: 26772746.

From the abstract: "Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate. Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone. In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. ... This atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly."

Monday, February 8, 2016

in vivo RNAi screen related to chromatin remodeling and assembly factor (CHD1)

Kim S, Bugga L, Hong ES, Zabinsky R, Edwards RG, Deodhar PA, Armstrong JA. An RNAi-Based Candidate Screen for Modifiers of the CHD1 Chromatin Remodeler and Assembly Factor in Drosophila melanogaster. G3 (Bethesda). 2015 Nov 23;6(2):245-54. PMID: 26596648.

From the abstract: "The conserved chromatin remodeling and assembly factor CHD1 (chromodomains, helicase, DNA-binding domain) is present at active genes where it participates in histone turnover and recycling during transcription. In order to gain a more complete understanding of the mechanism of action of CHD1 during development, we created a novel genetic assay in Drosophila melanogaster to evaluate potential functional interactions between CHD1 and other chromatin factors. We found that overexpression of CHD1 results in defects in wing development and utilized this fully penetrant and reliable phenotype to conduct a small-scale RNAi-based candidate screen ..."

Saturday, January 23, 2016

Tuesday, January 19, 2016

Intriguing new potential Alzheimer's biomarker test based on response by engineered Drosophila cells

Lau HC, Lee IK, Ko PW, Lee HW, Huh JS, Cho WJ, Lim JO. Non-invasive screening for Alzheimer's disease by sensing salivary sugar using Drosophila cells expressing gustatory receptor (Gr5a) immobilized on an extended gate ion-sensitive field-effect transistor (EG-ISFET) biosensor. PLoS One. 2015 Feb 25;10(2):e0117810. PMID: 25714733; PMCID: PMC4340960.

From the abstract:  "Body fluids are often used as specimens for medical diagnosis. With the advent of advanced analytical techniques in biotechnology, the diagnostic potential of saliva has been the focus of many studies. We recently reported the presence of excess salivary sugars, in patients with Alzheimer's disease (AD). In the present study, we developed a highly sensitive, cell-based biosensor to detect trehalose levels in patient saliva. The developed biosensor relies on the overexpression of sugar sensitive gustatory receptors (Gr5a) in Drosophila cells to detect the salivary trehalose. ... "

Thursday, January 14, 2016

Another paper reporting concern about an RNAi fly library

Any of us using the relevant RNAi stocks should be aware.

Vissers JH, Manning SA, Kulkarni A, Harvey KF. A Drosophila RNAi library modulates Hippo pathway-dependent tissue growth. Nat Commun. 2016 Jan 13;7:10368. PMID: 26758424.
From the abstract: "... Here we investigate an important technical limitation with the widely used VDRC KK RNAi collection. We find that approximately 25% of VDRC KK RNAi lines cause false-positive enhancement of the Hippo pathway, owing to ectopic expression of the Tiptop transcription factor. Of relevance to the broader Drosophila community, ectopic tiptop (tio) expression can also cause organ malformations and mask phenotypes such as organ overgrowth. To enhance the use of the VDRC KK RNAi library, we have generated a D. melanogaster strain that will allow researchers to test, in a single cross, whether their genetic screen of interest will be affected by ectopic tio expression."

Tuesday, January 12, 2016

Friday, January 8, 2016

Searched "Drosophila" at DSHB antibody hybridoma bank? Try again!

Rumor has it DSHB has updated search rules such that a search with the term "Drosophila" that used to bring back about 100 results now retrieves more than 200. Happy antibody hunting!

Tuesday, January 5, 2016

Fly cell RNAi screen identifies "first in vivo mediator of hypercapnic immune suppression" in study relevant to COPD and other lung disorders

Congrats to Taneli and the rest of the team on this report of a screen performed at the DRSC!

Helenius IT, Haake RJ, Kwon YJ, Hu JA, Krupinski T, Casalino-Matsuda SM, Sporn PH, Sznajder JI, Beitel GJ. Identification of Drosophila Zfh2 as a Mediator of Hypercapnic Immune Regulation by a Genome-Wide RNA Interference Screen. J Immunol. 2015 Dec 7. pii: 1501708. PMID: 26643480.

From the abstract: "Hypercapnia, elevated partial pressure of CO2 in blood and tissue, develops in many patients with chronic severe obstructive pulmonary disease and other advanced lung disorders. Patients with advanced disease frequently develop bacterial lung infections ... We previously demonstrated that hypercapnia suppresses induction of NF-κB-regulated innate immune response genes ... However, the molecular mediators of hypercapnic immune suppression are undefined. In this study, we report a genome-wide RNA interference screen in Drosophila S2* cells stimulated with bacterial peptidoglycan. The screen identified 16 genes with human orthologs whose knockdown reduced hypercapnic suppression of the gene encoding the antimicrobial peptide Diptericin (Dipt), but did not increase Dipt mRNA levels in air. In vivo tests of one of the strongest screen hits, zinc finger homeodomain 2 (Zfh2; mammalian orthologs ZFHX3/ATBF1 and ZFHX4), demonstrate that reducing zfh2 function using a mutation or RNA interference improves survival of flies exposed to elevated CO2 and infected with Staphylococcus aureus. Tissue-specific knockdown of zfh2 in the fat body, the major immune and metabolic organ of the fly, mitigates hypercapnia-induced reductions ... and improves resistance of CO2-exposed flies to infection. Zfh2 mutations also partially rescue hypercapnia-induced delays in egg hatching ... Taken together, to our knowledge, these results identify Zfh2 as the first in vivo mediator of hypercapnic immune suppression."