Wednesday, November 19, 2014

Review and detailed protocols--CRISPR-Cas9 in flies and fly cells

Housden BE, Lin S, Perrimon N. Cas9-based genome editing in Drosophila. Methods Enzymol. 2014;546:415-39. PMID: 25398351.  

From the abstract: "... we first discuss some general design principles for genome engineering experiments in Drosophila and then present detailed protocols for the production of CRISPR reagents and screening strategies to detect successful genome modification events in both tissue culture cells and animals."

Includes helpful tables listing sgRNA design tools, relevant fly stocks and plasmids.

Friday, November 14, 2014

Cross-species cell-based study of the lipoprotein lipase-binding protein GPIHBP1

Beigneux AP, Fong LG, Bensadoun A, Davies BS, Oberer M, Gårdsvoll H, Ploug M, Young SG. GPIHBP1 Missense Mutations Often Cause Multimerization of GPIHBP1 and Thereby Prevent Lipoprotein Lipase Binding. Circ Res. 2014 Nov 11. pii: CIRCRESAHA.114.305085. PMID: 25387803.

From the abstract: "GPIHBP1, a GPI-anchored protein of capillary endothelial cells, binds lipoprotein lipase (LPL) in the subendothelial spaces and shuttles it to the capillary lumen. GPIHBP1 missense mutations that interfere with LPL binding cause familial chylomicronemia. ... We expressed mutant forms of GPIHBP1 in Chinese hamster ovary cells, rat and human endothelial cells, and Drosophila S2 cells. In each expression system, mutation of cysteines in GPIHBP1's Ly6 domain (including mutants identified in chylomicronemia patients) led to the formation of disulfide-linked dimers and multimers. ..."

Tuesday, November 11, 2014

in vivo RNAi screen related to ALS

Deivasigamani S, Verma HK, Ueda R, Ratnaparkhi A, Ratnaparkhi GS. A genetic screen identifies Tor as an interactor of VAPB in a Drosophila model of amyotrophic lateral sclerosis. Biol Open. 2014 Oct 31. pii: BIO201410066. PMID: 25361581.

From the abstract: "Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by selective death of motor neurons. In 5-10% of the familial cases, the disease is inherited because of mutations. One such mutation, P56S, was identified in human VAPB that behaves in a dominant negative manner, sequestering wild type protein into cytoplasmic inclusions. We have conducted a reverse genetic screen to identify interactors of Drosophila VAPB. We screened 2635 genes and identified 103 interactors, of which 45 were enhancers and 58 were suppressors of VAPB function. Interestingly, the screen identified known ALS loci - TBPH, alsin2 and SOD1. Also identified were genes involved in cellular energetics and homeostasis which were used to build a gene regulatory network of VAPB modifiers. One key modifier identified was Tor, ..."

The biology of RNAi--recent studies

Bronkhorst AW, van Cleef KW, Venselaar H, van Rij RP. A dsRNA-binding protein of a complex invertebrate DNA virus suppresses the Drosophila RNAi response. Nucleic Acids Res. 2014 Oct 29;42(19):12237-48. PMID: 25274730.

From the abstract: "... Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R. ... Together, our findings indicate that, in analogy to RNA viruses, DNA viruses antagonize the antiviral RNAi response."

Gandhi SG, Bag I, Sengupta S, Pal-Bhadra M, Bhadra U. Drosophila oncogene Gas41 is RNAi modulator that intersects heterochromatin and siRNA pathway. FEBS J. 2014 Oct 16. PMID: 25323651.

From the abstract: "... These findings suggest that, Drosophila Gas41 guides the repeat associated gene silencing, and Dicer1 interaction thereby depicting a new role of the Gas41. ... In Drosophila, Gas41 plays a dual role. In one hand, it seems to participate with Dicer 1 in the RNAi pathway and alternatively also participate in repeat-induced gene silencing by accumulating heterochromatin proteins at the mw array promoters. ..."

Cell-based assays--new reports

Ramdas NM, Shivashankar GV. Cytoskeletal Control of Nuclear Morphology and Chromatin Organization. J Mol Biol. 2014 Oct 2. pii: S0022-2836(14)00495-1. PMID: 25281900.

From the abstract: "... We demonstrate here the differential influence of perinuclear actin- and microtubule-driven assemblies on nuclear architecture using pharmacological inhibitors and targeted RNA interference knockdown of cytoskeleton components in Drosophila cells. We find evidence that the loss of perinuclear actin assembly results in basolateral enhancement of microtubule organization and this is reflected functionally by enhanced nuclear dynamics. ..."

Ribeiro SA, D'Ambrosio MV, Vale RD. Induction of Focal Adhesions and Motility in Drosophila S2 cells. Mol Biol Cell. 2014 PMID: 25273555.

From the abstract: "... Here, we describe a system for inducing the formation of focal adhesions in normally non-ECM-adherent, non-motile Drosophila S2 cells. These focal adhesions contain the expected molecular markers such as talin, vinculin, and p130Cas, and they require talin for their formation. The S2 cells with induced focal adhesions also display a non-polarized form of motility on vitronectin-coated substrates. Consistent with findings in mammalian cells, the degree of motility can be tuned by changing the stiffness of the substrate and was increased after the depletion of PAK3, a p21-activated kinase. ... These results demonstrate that S2 cells, a cell line that is well studied for cytoskeletal dynamics and readily amenable to protein manipulation by RNAi, can be used to study the assembly and dynamics of focal adhesions and mechanosensitive cell motility."

Genome-wide RNAi screen related to Parkinson's disease

Ivatt RM, Sanchez-Martinez A, Godena VK, Brown S, Ziviani E, Whitworth AJ. Genome-wide RNAi screen identifies the Parkinson disease GWAS risk locus SREBF1 as a regulator of mitophagy. Proc Natl Acad Sci U S A. 2014 Jun 10;111(23):8494-9. PMID: 24912190; PMCID: PMC4060696.

From the abstract: "Genetic analysis of Parkinson disease (PD) has identified several genes whose mutation causes inherited parkinsonism, as well as risk loci for sporadic PD. PTEN-induced kinase 1 (PINK1) and parkin, linked to autosomal recessive PD, act in a common genetic pathway regulating the autophagic degradation of mitochondria, termed mitophagy. We undertook a genome-wide RNAi screen as an unbiased approach to identify genes regulating the PINK1/Parkin pathway. We identified several genes that have a conserved function in promoting mitochondrial translocation of Parkin and subsequent mitophagy, most notably sterol regulatory element binding transcription factor 1 (SREBF1), F-box and WD40 domain protein 7 (FBXW7), and other components of the lipogenesis pathway. ..."

in vivo genome-wide screen--NURF and dREAM

Bohla D, Herold M, Panzer I, Buxa MK, Ali T, Demmers J, Krüger M, Scharfe M, Jarek M, Bartkuhn M, Renkawitz R. A Functional Insulator Screen Identifies NURF and dREAM Components to Be Required for Enhancer-Blocking. PLoS One. 2014 Sep 23;9(9):e107765. PMID: 25247414; PMCID: PMC4172637.

From the abstract: "... We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident ..."