Tuesday, March 6, 2018

FlyBook review on RNAi screening in Drosophila cells and in vivo

Heigwer F, Port F, Boutros M. RNA Interference (RNAi) Screening in Drosophila. Genetics. 2018 Mar;208(3):853-874. PMID: 29487145.

From the abstract: "... RNA interference (RNAi) ... has had an important impact on identifying and characterizing gene function. First discovered in Caenorhabditis elegans, RNAi can be used to silence the expression of genes through introduction of exogenous double-stranded RNA into cells. In Drosophila, RNAi has been applied in cultured cells or in vivo to perturb the function of single genes or to systematically probe gene function on a genome-wide scale. In this review, we will describe the use of RNAi to study gene function in Drosophila with a particular focus on high-throughput screening methods applied in cultured cells. ..."

Friday, February 23, 2018

Fly iPCs? Opinion piece in Genes suggests it should be tried

Kaur P, Jin HJ, Lusk JB, Tolwinski NS. Modeling the Role of Wnt Signaling in Human and Drosophila Stem Cells. Genes (Basel). 2018 Feb 16;9(2). pii: E101. PMID: 29462894.

From the abstract: "The discovery of induced pluripotent stem (iPS) cells ... dramatically transformed the study of stem cells ... Although advances have pushed the field forward, human application remains some years away, in part due to the need for an in-depth mechanistic understanding. The role of Wnts in stem cells predates the discovery of iPS cells with Wnts established as major pluripotency promoting factors. Most work to date has been done using mouse and tissue culture models and few attempts have been made in other model organisms, but the recent combination of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing with iPS cell technology provides a perfect avenue for exploring iPS cells in model organisms. ... In this opinion article, we draw parallels between Wnt signaling in human and Drosophila stem cell systems, propose ways to obtain Drosophila iPS cells, and suggest ways to exploit the versatility of the Drosophila system for future stem cell studies."

Of related interest: RasV12 method for isolation of new cell lines, developed by A. Simcox (OSU).
http://flyrnai.blogspot.com/2012/10/breaking-report-cell-culture.html
http://flyrnai.blogspot.com/2009/01/technical-note-making-new-cell-lines.html

Wednesday, January 3, 2018

Single-molecule imaging study of a Wnt ligand using S2 and S2R+ Drosophila cultured cells

Lippert A, Janeczek AA, F├╝rstenberg A, Ponjavic A, Moerner WE, Nusse R, Helms JA, Evans ND, Lee SF. Single-Molecule Imaging of Wnt3A Protein Diffusion on Living Cell Membranes. Biophys J. 2017 Dec 19;113(12):2762-2767. PMID: 29262368.

Abstract: "Wnt proteins are secreted, hydrophobic, lipidated proteins found in all animals that play essential roles in development and disease. Lipid modification is thought to facilitate the interaction of the protein with its receptor, Frizzled, but may also regulate the transport of Wnt protein and its localization at the cell membrane. Here, by employing single-molecule fluorescence techniques, we show that Wnt proteins associate with and diffuse on the plasma membranes of living cells in the absence of any receptor binding. We find that labeled Wnt3A transiently and dynamically associates with the membranes of Drosophila Schneider 2 cells, diffuses with Brownian kinetics on flattened membranes and on cellular protrusions, and does not transfer between cells in close contact. In S2 receptor-plus (S2R+) cells, which express Frizzled receptors, membrane diffusion rate is reduced and membrane residency time is increased. These results provide direct evidence of Wnt3A interaction with living cell membranes, and represent, to our knowledge, a new system for investigating the dynamics of Wnt transport."

Wednesday, December 13, 2017

DRSC screen contributes to research report on signaling and lifespan

Sung EJ, Ryuda M, Matsumoto H, Uryu O, Ochiai M, Cook ME, Yi NY, Wang H, Putney JW, Bird GS, Shears SB, Hayakawa Y. Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress. Proc Natl Acad Sci U S A. 2017 Dec 11. PMID: 29229844.

From the abstract: "A systems-level understanding of cytokine-mediated, intertissue signaling is one of the keys to developing fundamental insight into the links between aging and inflammation. Here, we employed Drosophila, a routine model for analysis of cytokine signaling pathways in higher animals, to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca2+ signaling pathway mediates innate immune responses to GBP, we conducted a dsRNA library screen for genes that modulate Ca2+ mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. We discovered that the metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This we established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. ... We describe how our data offer opportunities for further molecular interrogation of yin and yang between homeostasis and longevity."

Monday, December 11, 2017

RNAi and the sterile insect technique

Darrington M, Dalmay T, Morrison NI, Chapman T. Implementing the sterile insect technique with RNA interference - a review. Entomol Exp Appl. 2017 Sep;164(3):155-175. PMID: 29200471; PMCID: PMC5697603.

From the abstract: "We review RNA interference (RNAi) of insect pests and its potential for implementing sterile insect technique (SIT)-related control. The molecular mechanisms that support RNAi in pest species are reviewed in detail, drawing on literature from a range of species including Drosophila melanogaster Meigen and Homo sapiens L. ... RNAi represents a plausible, non-GM system for targeting populations of insects for control purposes, if RNAi effector molecules can be delivered environmentally (eRNAi). We consider studies of eRNAi from across several insect orders and review to what extent taxonomy, genetics, and differing methods of double-stranded (ds) RNA synthesis and delivery can influence the efficiency of gene knockdown. ..."

Monday, December 4, 2017

New report on qPCR design for RNAi knockdown effectiveness testing

Mainland RL, Lyons TA, Ruth MM, Kramer JM. Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines. BMC Res Notes. 2017 Nov 29;10(1):647. PMID: 29187229.

From the abstract: "... the strongest gene knockdown was detected when qPCR was performed using 5' primer sets in combination with mRNA-derived cDNA. Our results indicate that detection of undegraded mRNA cleavage fragments can result in underestimation of true knockdown efficiency for a RNAi construct. ..."

Wednesday, July 19, 2017

Short vs. long hairpins in fly RNAi

Bartoletti R, Capozzoli B, Moore J, Moran J, Shrawder B, Vivekanand P. Short hairpin RNA is more effective than long hairpin RNA in eliciting pointed loss-of-function phenotypes in Drosophila. Genesis. 2017 Jul;55(7). PMID: 28464429.

From the abstract: "Pointed (Pnt) is a transcriptional activator ... As mutations in pnt perturb the development of several tissues, we wanted to examine the effect and efficacy of using RNAi to target Pnt. We have expressed pnt RNAi in the eyes, oocyte, and heart cells using three different RNAi lines: Valium20, Valium10, and VDRC. Valium20 is distinct since it generates a short hairpin RNA (shRNA), while Valium10 and VDRC produce long hairpin dsRNA. We found that for each tissue examined Valium20 exhibited the strongest phenotype while the Valium10 and VDRC lines produced varying levels of severity and that the long hairpin RNA produced by the Valium10 and VDRC lines are unable to effectively knockdown pnt in embryonic tissues."