in vivo RNAi screen explores tendons

Tiwari P, Kumar A, Das RN, Malhotra V, VijayRaghavan K. A Tendon Cell Specific RNAi Screen Reveals Novel Candidates Essential for Muscle Tendon Interaction. PLoS One. 2015 Oct 21;10(10):e0140976. PMID: 26488612.

From the abstract: "... We performed a genetic screen using RNAi-mediated knockdown in tendon cells to find out molecular players involved in the formation and maintenance of myotendinous junction and found 21 candidates out of 2507 RNAi lines screened. Of these, 19 were novel molecules in context of myotendinous system. ..."

Review by Venken and colleagues on genome engineering in flies

Venken KJ, Sarrion-Perdigones A, Vandeventer PJ, Abel NS, Christiansen AE, Hoffman KL. Genome engineering: Drosophila melanogaster and beyond. Wiley Interdiscip Rev Dev Biol. 2015 Oct 8. PMID: 26447401.

From the abstract: "Here, we summarize different ways to perform precise inheritable genome engineering using integrases, recombinases, and DNA nucleases in the D. melanogaster."

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."

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:

• Single-cell cloning of Drosophila cells
• Making stably transfected Drosophila cells

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.

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. ..."