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."