Kunzelmann S, Böttcher R, Schmidts I, Förstemann K. A Comprehensive Toolbox for Genome Editing in Cultured Drosophila melanogaster Cells. G3 (Bethesda). 2016 Jun 1;6(6):1777-85. PMID: 27172193
From the abstract: "... Following up on our initial publication, we now describe a considerably simplified, more efficient, and readily scalable experimental workflow for PCR-based genome editing in cultured Drosophila melanogaster cells. Our analysis at the act5C locus suggests that PCR-based homology arms of 60 bp are sufficient to reach targeting efficiencies of up to 80% after selection; extension to 80 bp (PCR) or 500 bp (targeting vector) did not further improve the yield. We have expanded our targeting system to N-terminal epitope tags; this also allows the generation of cell populations with heterologous expression control of the tagged locus via the copper-inducible mtnDE promoter. We present detailed, quantitative data on editing efficiencies for several genomic loci that may serve as positive controls or benchmarks in other laboratories. ..."
Thursday, June 30, 2016
EP-MS2 method and RNAi follow-up used in screen for localized RNAs
Misra M, Edmund H, Ennis D, Schlueter MA, Marot JE, Tambasco J, Barlow I, Sigurbjornsdottir S, Mathew R, Vallés AM, Davis I, Leptin M, Gavis ER. A Genome-Wide Screen for Dendritically Localized RNAs Identifies Genes Required for Dendrite Morphogenesis. G3 (Bethesda). 2016 Jun 3. pii: g3.116.030353. PMID: 27260999.
From the abstract: "Localizing messenger RNAs at specific subcellular sites is a conserved mechanism for targeting the synthesis of cytoplasmic proteins to distinct subcellular domains ... However, the full range of transcripts that are asymmetrically distributed in specialized cell types and the significance of their localization, especially in the nervous system, are not known. We used the EP-MS2 method, which combines EP transposon insertion with the MS2/MCP in vivo fluorescent labeling system to screen for novel localized transcripts in polarized cells, focusing on the highly branched Drosophila class IV dendritic arborization neurons. Of a total of 541 lines screened, we identified 55 EP-MS2 insertions producing transcripts that were enriched in neuronal processes, particularly in dendrites. The 47 genes identified by these insertions encode molecularly diverse proteins and are enriched for genes that function in neuronal development and physiology. RNAi-mediated knockdown confirmed roles for many of the candidate genes in dendrite morphogenesis. ..."
From the abstract: "Localizing messenger RNAs at specific subcellular sites is a conserved mechanism for targeting the synthesis of cytoplasmic proteins to distinct subcellular domains ... However, the full range of transcripts that are asymmetrically distributed in specialized cell types and the significance of their localization, especially in the nervous system, are not known. We used the EP-MS2 method, which combines EP transposon insertion with the MS2/MCP in vivo fluorescent labeling system to screen for novel localized transcripts in polarized cells, focusing on the highly branched Drosophila class IV dendritic arborization neurons. Of a total of 541 lines screened, we identified 55 EP-MS2 insertions producing transcripts that were enriched in neuronal processes, particularly in dendrites. The 47 genes identified by these insertions encode molecularly diverse proteins and are enriched for genes that function in neuronal development and physiology. RNAi-mediated knockdown confirmed roles for many of the candidate genes in dendrite morphogenesis. ..."
Tuesday, June 28, 2016
in vivo RNAi screen related to cholesterol and steroid hormones
Danielsen ET, Moeller ME, Yamanaka N, Ou Q, Laursen JM, Soenderholm C, Zhuo R, Phelps B, Tang K, Zeng J, Kondo S, Nielsen CH, Harvald EB, Faergeman NJ, Haley MJ, O'Connor KA, King-Jones K, O'Connor MB, Rewitz KF. A Drosophila Genome-Wide Screen Identifies Regulators of Steroid Hormone Production and Developmental Timing. Dev Cell. 2016 Jun 20;37(6):558-70. PMID: 27326933; PMCID: PMC4918455.
From the abstract: "Steroid hormones control important developmental processes and are linked to many diseases. To systematically identify genes and pathways required for steroid production, we performed a Drosophila genome-wide in vivo RNAi screen and identified 1,906 genes with potential roles in steroidogenesis and developmental timing. Here, we use our screen as a resource to identify mechanisms regulating intracellular levels of cholesterol, a substrate for steroidogenesis. ... In addition, we demonstrate the existence of an autophagosomal cholesterol mobilization mechanism and show that activation of this system rescues Niemann-Pick type C1 deficiency that causes a disorder characterized by cholesterol accumulation. ... These results reveal genes regulating steroidogenesis during development that likely modulate disease mechanisms."
From the abstract: "Steroid hormones control important developmental processes and are linked to many diseases. To systematically identify genes and pathways required for steroid production, we performed a Drosophila genome-wide in vivo RNAi screen and identified 1,906 genes with potential roles in steroidogenesis and developmental timing. Here, we use our screen as a resource to identify mechanisms regulating intracellular levels of cholesterol, a substrate for steroidogenesis. ... In addition, we demonstrate the existence of an autophagosomal cholesterol mobilization mechanism and show that activation of this system rescues Niemann-Pick type C1 deficiency that causes a disorder characterized by cholesterol accumulation. ... These results reveal genes regulating steroidogenesis during development that likely modulate disease mechanisms."
Friday, June 10, 2016
Combined CRISPR-RNAi approach
On my desk to read today? This report from former Perrimonian and DRSC screener R. Neumuller:
Wissel S, Kieser A, Yasugi T, Duchek P, Roitinger E, Gokcezade JF, Steinmann V, Gaul U, Mechtler K, Förstemann K, Knoblich JA, Neumüller RA. A Combination of CRISPR/Cas9 and Standardized RNAi as a Versatile Platform for the Characterization of Gene Function. G3 (Bethesda). 2016 Jun 8. pii: g3.116.028571. PMID: 27280787.
From the abstract: "... miGFPi combines CRISPR/Cas9 mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method ... In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. ..."
Wissel S, Kieser A, Yasugi T, Duchek P, Roitinger E, Gokcezade JF, Steinmann V, Gaul U, Mechtler K, Förstemann K, Knoblich JA, Neumüller RA. A Combination of CRISPR/Cas9 and Standardized RNAi as a Versatile Platform for the Characterization of Gene Function. G3 (Bethesda). 2016 Jun 8. pii: g3.116.028571. PMID: 27280787.
From the abstract: "... miGFPi combines CRISPR/Cas9 mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method ... In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. ..."
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