Friday, October 9, 2015

DGRC team reports on targeted insertion approach to modifying Drosophila cultured cell lines

Lucy Cherbas, Jennifer Hackney, Lei Gong, Claire Salzer, Eric Mauser, Dayu Zhang and Peter Cherbas. 2015. Tools for Targeted Genome Engineering of Established Drosophila Cell Lines. Early online at Genetics.

From the abstract: "We describe an adaptation of φC31 integrase-mediated targeted cassette exchange for use in Drosophila cell lines. ... We demonstrated the technology by integrating a cassette containing a Cu++-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays – a major emphasis of cell-based studies."

Tuesday, October 6, 2015

New at the DRSC website--protocols for single-cell cloning and stable transfection of Drosophila cells

Two new step-by-step protocols available at the DRSC website:

And check out these related publications and resources:

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

Housden BE, Valvezan AJ, Kelley C, Sopko R, Hu Y, Roesel C,
Lin S, Buckner M, Tao R, Yilmazel B, Mohr SE, Manning BD, Perrimon N. Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. Sci Signal. 2015 Sep 8;8(393):rs9. PMID: 26350902.

Santos MG, Jorge SA, Brillet K, Pereira CA. Improving heterologous protein expression in transfected Drosophila S2 cells as assessed by EGFP expression. Cytotechnology. 2007 May;54(1):15-24. PMID: 19003014; PMCID: PMC2267513.

Have a qPCR machine but no software for high-resolution melt analysis following CRISPR modification? Check out the HRMA online tool.

Monday, October 5, 2015

Methods review on genome-wide cell-based screens to identify centrosome components

Dobbelaere J. Genome-wide RNAi screens in S2 cells to identify centrosome components. Methods Cell Biol. 2015;129:279-300. PMID: 26175444.

From the abstract: "... In this paper, we present detailed instructions for designing, performing, and analyzing a genome-wide screen in Drosophila tissue culture cells to identify centrosome components using a microscopy-based approach. "

"ex vivo" fly RNAi screen

Kanoh H, Kuraishi T, Tong LL, Watanabe R, Nagata S, Kurata S. Ex vivo genome-wide RNAi screening of the Drosophila Toll signaling pathway elicited by a larva-derived tissue extract. Biochem Biophys Res Commun. 2015 Sep 28. pii: S0006-291X(15)30654-9. PMID: 26427875.

From the abstract: "... In Drosophila, the Toll pathway confers damage responses during bacterial infection and improper cell-fate control. However, the intrinsic ligands and signaling mechanisms that potentiate innate immune responses remain unknown. Here, we demonstrate that a Drosophila larva-derived tissue extract strongly elicits Toll pathway activation via the Toll receptor. Using this extract, we performed ex vivo genome-wide RNAi screening in Drosophila cultured cells, and identified several signaling factors that are required for host defense and antimicrobial-peptide expression in Drosophila adults. ..."

Tuesday, September 29, 2015

One-generation in vivo Drosophila genetic screening approach relevant to rhabdomyosarcoma

Galindo KA, Endicott TR, Avirneni-Vadlamudi U, Galindo RL. A rapid one-generation genetic screen in a Drosophila model to capture rhabdomyosarcoma effectors and therapeutic targets. G3 (Bethesda). 2014 Dec 9;5(2):205-17. PMID: 25491943; PMCID: PMC4321029.

From the abstract: "Rhabdomyosarcoma (RMS) is an aggressive childhood malignancy of neoplastic muscle-lineage precursors that fail to terminally differentiate into syncytial muscle. The most aggressive form of RMS, alveolar-RMS, is driven by misexpression of the PAX-FOXO1 oncoprotein ... Here, we report a new approach to dissect RMS, exploiting a highly efficient Drosophila PAX7-FOXO1 model uniquely configured to uncover PAX-FOXO1 RMS genetic effectors in only one generation. ... These studies show the utility of the PAX-FOXO1 Drosophila system as a robust one-generation (F1) RMS gene discovery platform and demonstrate how Drosophila transgenic conditional expression models can be configured for the rapid dissection of human disease. "

Monday, September 28, 2015

RNA-based mitochondrial targeting technique shown to work in vivo in flies and in human cells

Towheed A, Markantone DM, Crain AT, Celotto AM, Palladino MJ. Small mitochondrial-targeted RNAs modulate endogenous mitochondrial protein expression in vivo. Neurobiol Dis. 2014 Sep;69:15-22. PMID: 24807207; PMCID: PMC4106415.

From the abstract: “Endogenous mitochondrial genes encode critical oxidative phosphorylation components and their mutation results in a set of disorders known collectively as mitochondrial encephalomyopathies. There is intensive interest in modulating mitochondrial function as organelle dysfunction has been associated with numerous disease states. Proteins encoded by the mitochondrial genome cannot be genetically manipulated by current techniques. Here we report the development of a mitochondrial-targeted RNA expression system (mtTRES) utilizing distinct non-coding leader sequences (NCLs) and enabling in vivo expression [in Drosophila] of small mitochondrial-targeted RNAs. mtTRES expressing small chimeric antisense RNAs was used as translational inhibitors (TLIs) ... This technique has important and obvious research and clinical implications.”

Tuesday, September 22, 2015

Small ORF-related genome-wide screen performed at the DRSC reported by Zanet, Benrabah et al. in Science

Zanet J, Benrabah E, Li T, Pélissier-Monier A, Chanut-Delalande H, Ronsin B, Bellen HJ, Payre F, Plaza S. Pri sORF peptides induce selective proteasome-mediated protein processing. Science. 2015 Sep 18;349(6254):1356-8. PMID: 26383956.

From the abstract: "A wide variety of RNAs encode small open-reading-frame (smORF/sORF) peptides, but their functions are largely unknown. Here, we show that Drosophila polished-rice (pri) sORF peptides trigger proteasome-mediated protein processing, converting the Shavenbaby (Svb) transcription repressor into a shorter activator. A genome-wide RNA interference screen identifies an E2-E3 ubiquitin-conjugating complex, UbcD6-Ubr3, which targets Svb to the proteasome in a pri-dependent manner. ... "