Thursday, December 10, 2015
RNAi as insecticide
David O'Brachta at the IGT-RCN reports on insecticidal dsRNA encoded by corn, which is "moving to the field" currently "for seed production and breeding only." Read the story here.
Tuesday, December 1, 2015
Endogenous roles of Dicer-2--new report suggests link to Toll
Wang Z, Wu D, Liu Y, Xia X, Gong W, Qiu Y, Yang J, Zheng Y, Li J, Wang YF, Xiang Y, Hu Y, Zhou X. Drosophila Dicer-2 has an RNA interference-independent function that modulates Toll immune signaling. Sci Adv. 2015 Oct 16;1(9):e1500228. PMID: 26601278; PMCID: PMC4646792.
From the abstract: "Dicer-2 is the central player for small interfering RNA biogenesis in the Drosophila RNA interference (RNAi) pathway. Intriguingly, we found that Dicer-2 has an unconventional RNAi-independent function that positively modulates Toll immune signaling ... The loss of Dicer-2 expression makes fruit flies more susceptible to fungal infection. We further revealed that Dicer-2 posttranscriptionally modulates Toll signaling because Dicer-2 is required for the proper expression of Toll protein ... Dicer-2 directly binds to the 3' untranslated region (3'UTR) of Toll mRNA via its PAZ (Piwi/Argonaute/Zwille) domain and is required for protein translation mediated by Toll 3'UTR. ... our findings ... indicate an unexpected intersection of the RNAi pathway and the Toll pathway, and provide new insights into Toll immune signaling, Drosophila Dicer-2, and probably Dicer and Dicer-related proteins in other organisms."
From the abstract: "Dicer-2 is the central player for small interfering RNA biogenesis in the Drosophila RNA interference (RNAi) pathway. Intriguingly, we found that Dicer-2 has an unconventional RNAi-independent function that positively modulates Toll immune signaling ... The loss of Dicer-2 expression makes fruit flies more susceptible to fungal infection. We further revealed that Dicer-2 posttranscriptionally modulates Toll signaling because Dicer-2 is required for the proper expression of Toll protein ... Dicer-2 directly binds to the 3' untranslated region (3'UTR) of Toll mRNA via its PAZ (Piwi/Argonaute/Zwille) domain and is required for protein translation mediated by Toll 3'UTR. ... our findings ... indicate an unexpected intersection of the RNAi pathway and the Toll pathway, and provide new insights into Toll immune signaling, Drosophila Dicer-2, and probably Dicer and Dicer-related proteins in other organisms."
Tuesday, November 24, 2015
Drosophila cell team at the DGRC reports integrase-mediated cassette exchange approach to production of clonal transgenic fly cell lines
Lucy Cherbas1, Jennifer Hackney, Lei Gong, Claire Salzer, Eric Mauser, Dayu Zhang and Peter Cherbas.
Tools for Targeted Genome Engineering of Established Drosophila Cell Lines.
Early online in G3.
From the abstract: "We describe an adaptation of ΦC31 integrase-mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking site lines carries a single mapped copy of one of the docking platforms. ... We describe procedures for isolating cells carrying the substitutions ... When compared with clonal lines made by traditional transformation methods ... 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."
Early online in G3.
From the abstract: "We describe an adaptation of ΦC31 integrase-mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking site lines carries a single mapped copy of one of the docking platforms. ... We describe procedures for isolating cells carrying the substitutions ... When compared with clonal lines made by traditional transformation methods ... 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."
RNAi-based screen for interaction with chd1
Sharon Kim, Lakshmi Bugga, Eugenie S. Hong, Rebecca Zabinsky, Rebecca G. Edwards, Parimal A. Deodhar and Jennifer A. Armstrong. An RNAi-Based Candidate Screen for Modifiers of the CHD1 Chromatin Remodeler and Assembly Factor in Drosophila melanogaster.
Early online at G3.
From the abstract: "... CHD1 ... is present at active genes where it participates in histone turnover and recycling during transcription. ... We found that over-expression of the CHD1 results in defects in wing development and utilized this fully penetrant and reliable phenotype to conduct a small-scale RNAi-based candidate screen to identify genes that functionally interact with chd1 in vivo. ..."
Early online at G3.
From the abstract: "... CHD1 ... is present at active genes where it participates in histone turnover and recycling during transcription. ... We found that over-expression of the CHD1 results in defects in wing development and utilized this fully penetrant and reliable phenotype to conduct a small-scale RNAi-based candidate screen to identify genes that functionally interact with chd1 in vivo. ..."
Tuesday, November 17, 2015
in vivo RNAi used to validate at gene level hits in genome-wide deficiency screen for enhancers and suppressors of Na (+) /K (+) ATPase alleles
Talsma AD, Chaves JF, LaMonaca A, Wieczorek ED, Palladino MJ. Genome-wide screen for modifiers of Na (+) /K (+) ATPase alleles identifies critical genetic loci. Mol Brain. 2014 Dec 5;7:89. doi: 10.1186/s13041-014-0089-3. PMID: 25476251; PMCID: PMC4302446.
From the abstract: "Mutations affecting the Na (+) / K (+) ATPase (a.k.a. the sodium-potassium pump) genes cause conditional locomotor phenotypes in flies and three distinct complex neurological diseases in humans. More than 50 mutations have been identified affecting the human ATP1A2 and ATP1A3 genes that are known to cause rapid-onset Dystonia Parkinsonism, familial hemiplegic migraine, alternating hemiplegia of childhood, and variants of familial hemiplegic migraine with neurological complications including seizures and various mood disorders. In flies, mutations affecting the ATPalpha gene have dramatic phenotypes including altered longevity, neural dysfunction, neurodegeneration, myodegeneration, and striking locomotor impairment. ... We performed a genome-wide deficiency screen ... to identify novel modifier loci. A secondary screen confirmed allele-specificity of the interactions and many of the interactions were mapped to single genes and subsequently validated. We successfully identified 64 modifier loci and used classical mutations and RNAi to confirm 50 single gene interactions. ... These data demonstrate there are many loci capable of modifying ATPalpha dysfunction, which may provide the basis for modifying migraine, locomotor and seizure dysfunction in animals."
From the abstract: "Mutations affecting the Na (+) / K (+) ATPase (a.k.a. the sodium-potassium pump) genes cause conditional locomotor phenotypes in flies and three distinct complex neurological diseases in humans. More than 50 mutations have been identified affecting the human ATP1A2 and ATP1A3 genes that are known to cause rapid-onset Dystonia Parkinsonism, familial hemiplegic migraine, alternating hemiplegia of childhood, and variants of familial hemiplegic migraine with neurological complications including seizures and various mood disorders. In flies, mutations affecting the ATPalpha gene have dramatic phenotypes including altered longevity, neural dysfunction, neurodegeneration, myodegeneration, and striking locomotor impairment. ... We performed a genome-wide deficiency screen ... to identify novel modifier loci. A secondary screen confirmed allele-specificity of the interactions and many of the interactions were mapped to single genes and subsequently validated. We successfully identified 64 modifier loci and used classical mutations and RNAi to confirm 50 single gene interactions. ... These data demonstrate there are many loci capable of modifying ATPalpha dysfunction, which may provide the basis for modifying migraine, locomotor and seizure dysfunction in animals."
Monday, October 26, 2015
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. ..."
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. ..."
Wednesday, October 14, 2015
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."
From the abstract: "Here, we summarize different ways to perform precise inheritable genome engineering using integrases, recombinases, and DNA nucleases in the D. melanogaster."
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."
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.
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. "
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. ..."
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. "
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.”
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. ... "
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. ... "
Monday, September 14, 2015
S2 cells used to express human HER2 protein domains
Kanthala S, Mill CP, Riese DJ 2nd, Jaiswal M, Jois S. Expression And Purification Of HER2 Extracellular Domain Proteins In Schneider2 Insect Cells. Protein Expr Purif. 2015 Sep 9. PMID: 26363121.
CRISPR + RNAi screening in Drosophila cells points to potential new drug targets for treatment of tuberous sclerosis complex (TSC)
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.
From the abstract: "The tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex ... Mutation or aberrant inhibition of the TSC complex is common in various human tumor syndromes and cancers. The discovery of novel therapeutic strategies to selectively target cells with functional loss of this complex is therefore of clinical relevance ... We developed a CRISPR-based method to generate homogeneous mutant Drosophila cell lines. By combining TSC1 or TSC2 mutant cell lines with RNAi screens against all kinases and phosphatases, we identified synthetic interactions with TSC1 and TSC2. Individual knockdown of three candidate genes ... reduced the population growth rate of Drosophila cells lacking either TSC1 or TSC2 but not that of wild-type cells ... [and] had similar growth-inhibiting effects in mammalian TSC2-deficient cell lines, including human tumor-derived cells, illustrating the power of this cross-species screening strategy to identify potential drug targets."
From the abstract: "The tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex ... Mutation or aberrant inhibition of the TSC complex is common in various human tumor syndromes and cancers. The discovery of novel therapeutic strategies to selectively target cells with functional loss of this complex is therefore of clinical relevance ... We developed a CRISPR-based method to generate homogeneous mutant Drosophila cell lines. By combining TSC1 or TSC2 mutant cell lines with RNAi screens against all kinases and phosphatases, we identified synthetic interactions with TSC1 and TSC2. Individual knockdown of three candidate genes ... reduced the population growth rate of Drosophila cells lacking either TSC1 or TSC2 but not that of wild-type cells ... [and] had similar growth-inhibiting effects in mammalian TSC2-deficient cell lines, including human tumor-derived cells, illustrating the power of this cross-species screening strategy to identify potential drug targets."
Monday, August 31, 2015
Resource paper on the TRiP fly stocks now published at Genetics
Transgenic RNAi Project (TRiP) RNAi fly & data resource paper now online. Congrats Liz & whole team! #GeneticsGSA http://t.co/xaHapyQUgl
— Stephanie Mohr (@smohrfly) August 31, 2015
Thursday, August 27, 2015
New resource--Enhancer trap flippase fly stocks
Smith BN, Ghazanfari AM, Bohm RA, Welch WP, Zhang B, Masly JP. A Flippase-Mediated GAL80/GAL4 Intersectional Resource for Dissecting Appendage Development in Drosophila. G3 (Bethesda). 2015 Aug 13. pii: g3.115.019810. PMID: 26276385.
From the abstract: "... A recently developed Flippase-induced intersectional GAL80/GAL4 repression method incorporates several gene manipulation technologies in Drosophila to enable such fine-scale dissection in neural tissues. ... Here, we expand the utility of a large collection of these enhancer-trap flippase transgenic insertion lines by characterizing their expression patterns in third larval instar imaginal discs. We screened 521 different enhancer-trap flippase lines and identified 28 that are expressed in imaginal tissues, including two transgenes that show sex-specific expression patterns. ... The results of our experiments show that these enhancer-trap flippase lines enable fine-scale manipulation in imaginal discs."
From the abstract: "... A recently developed Flippase-induced intersectional GAL80/GAL4 repression method incorporates several gene manipulation technologies in Drosophila to enable such fine-scale dissection in neural tissues. ... Here, we expand the utility of a large collection of these enhancer-trap flippase transgenic insertion lines by characterizing their expression patterns in third larval instar imaginal discs. We screened 521 different enhancer-trap flippase lines and identified 28 that are expressed in imaginal tissues, including two transgenes that show sex-specific expression patterns. ... The results of our experiments show that these enhancer-trap flippase lines enable fine-scale manipulation in imaginal discs."
Wednesday, August 19, 2015
DRSC FlyRNAi website will be down briefly on Sat. Aug. 22
FYI, Due to a central IT upgrade, the DRSC FlyRNAi website will be down Saturday August, 22, from 9:00 AM - 11:00 AM Eastern US time.
Thursday, August 6, 2015
CRISPR activation in vivo in Drosophila
Things are looking up: #CRISPR activation in vivo in #Drosophila. Early online in #Genetics. http://t.co/zojqKxmf2G
— Stephanie Mohr (@smohrfly) August 6, 2015Friday, July 31, 2015
CRISPR, gene drives and safety
Two recent papers speak to CRISPR, gene drives and safety:
Akbari et al. 2015 Safeguarding gene drive experiments in the laboratory. in Early online in Policy Forum section of Science.
Unckless et al. 2015 Modeling the Manipulation of Natural Populations by the Mutagenic Chain Reaction Early online at Genetics.
Akbari et al. 2015 Safeguarding gene drive experiments in the laboratory. in Early online in Policy Forum section of Science.
Abstract: "Multiple strategies are needed to ensure safe gene drive experiments."
Unckless et al. 2015 Modeling the Manipulation of Natural Populations by the Mutagenic Chain Reaction Early online at Genetics.
From the Discussion section: "... there are conditions in which accidental introductions of a single individual can lead to fixation of the MCR allele ..."
Wednesday, July 29, 2015
Beyond fruit flies--iBeetle large-scale RNAi screen in Tribolium
Schmitt-Engel C, Schultheis D, Schwirz J, Ströhlein N, Troelenberg N, Majumdar U, Dao VA, Grossmann D, Richter T, Tech M, Dönitz J, Gerischer L, Theis M, Schild I, Trauner J, Koniszewski ND, Küster E, Kittelmann S, Hu Y, Lehmann S, Siemanowski J, Ulrich J, Panfilio KA, Schröder R, Morgenstern B, Stanke M, Buchhholz F, Frasch M, Roth S, Wimmer EA, Schoppmeier M, Klingler M, Bucher G.
The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology. Nat Commun. 2015 Jul 28;6:7822. PMID: 26215380.
From the abstract: "Genetic screens are powerful tools to identify the genes required for a given biological process. .. for technical reasons, comprehensive screens have been restricted to very few model organisms. ... although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum ... This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila."
Don't miss: "New fields of research" section of the article, which describes studies, such as of the 'odiferous stink glands,' that can be done in Tribolium but not Drosophila.
From the abstract: "Genetic screens are powerful tools to identify the genes required for a given biological process. .. for technical reasons, comprehensive screens have been restricted to very few model organisms. ... although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum ... This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila."
Don't miss: "New fields of research" section of the article, which describes studies, such as of the 'odiferous stink glands,' that can be done in Tribolium but not Drosophila.
Monday, July 27, 2015
Glia-specific RNAi screen explores circadian behavior
Ng FS, Jackson FR. The ROP vesicle release factor is required in adult Drosophila glia for normal circadian behavior. Front Cell Neurosci. 2015 Jul 3;9:256. PMID: 26190976.
Drosophila in vivo RNAi screen explores ovarian follicle cell differentiation
Jia D, Soylemez M, Calvin G, Bornmann R, Bryant J, Hanna C, Huang YC, Deng WM. A large-scale in vivo RNAi screen to identify genes involved in Notch-mediated follicle cell differentiation and cell cycle switches. Sci Rep. 2015 Jul 24;5:12328. PMID: 26205122.
Drosophila CRISPR library reported by Bassett et al
Bassett AR, Kong L, Liu JL. A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells. J Genet Genomics. 2015 Jun 20;42(6):301-9. PMID: 26165496.
Wednesday, July 22, 2015
DRSC publishes RBP library resource including 'baseline data' available lots of ways--including interactive!
The DRSC is pleased to announce our new cell-based RNAi library targeting RNA Binding Proteins and corresponding 'baseline' data set. These data should be a helpful reference for anyone analyzing data from screens of the same library with more sophisticated assays.
Big thanks to all the folks who worked hard to make this possible!
Mohr, Hu, Rudd, Buckner, Gilly, Foster, Sierzputowska, Comjean, Ye and Perrimon (2015) Reagent and Data Resources for Investigation of RNA Binding Protein Functions in Drosophila melanogaster Cultured Cells. Early online at G3 July 2015.
At a DRSC webpage we summarize ways to view the data.
Just one example--when you view the total ATP readout data at Plot.ly you can hover to see gene and reagent identifiers, zoom in on sub-sections of the graph, etc. The data are also available at the DRSC, at NCBI PubChem and no doubt will be imported by the folks at GenomeRNAi.
Big thanks to all the folks who worked hard to make this possible!
Mohr, Hu, Rudd, Buckner, Gilly, Foster, Sierzputowska, Comjean, Ye and Perrimon (2015) Reagent and Data Resources for Investigation of RNA Binding Protein Functions in Drosophila melanogaster Cultured Cells. Early online at G3 July 2015.
At a DRSC webpage we summarize ways to view the data.
Just one example--when you view the total ATP readout data at Plot.ly you can hover to see gene and reagent identifiers, zoom in on sub-sections of the graph, etc. The data are also available at the DRSC, at NCBI PubChem and no doubt will be imported by the folks at GenomeRNAi.
New report on TALEN gene editing in flies
An optimized TALEN application for mutagenesis and screening in Drosophila melanogaster. http://t.co/zcvHP87UWl
— flypapers (@fly_papers) July 22, 2015
Monday, July 20, 2015
S2 cell-based assay related to bioactive lipids
Wang P, Wang Q, Yang L, Qin QL, Wu YJ. Characterization of lysophosphatidylcholine-induced changes of intracellular calcium in Drosophila S2 cells. Life Sci. 2015 Jun 15;131:57-62. PMID: 25736969.
From the abstract: "Lysophosphatidylcholine (LPC), a bioactive lipid, regulates a wide array of biological processes. ... We aimed to study the mechanism of LPC-induced [Ca(2+)]i changes in Drosophila S2 cells. ... The [Ca(2+)]i of Drosophila S2 cells was measured by fluorescence spectrophotometry after loading the cells with the calcium-sensitive fluorescent probe Fura-2/AM. ...
Our results demonstrated that LPC could cause a rapid, dose-dependent increase in the [Ca(2+)]i in the presence of external calcium ([Ca(2+)]e). ... The LPC-induced [Ca(2+)]i increase in S2 cells characterized in this study may shed light on the study of NTE/SWS protein function in general because the enzyme is responsible for the deacylation of LPC."
From the abstract: "Lysophosphatidylcholine (LPC), a bioactive lipid, regulates a wide array of biological processes. ... We aimed to study the mechanism of LPC-induced [Ca(2+)]i changes in Drosophila S2 cells. ... The [Ca(2+)]i of Drosophila S2 cells was measured by fluorescence spectrophotometry after loading the cells with the calcium-sensitive fluorescent probe Fura-2/AM. ...
Our results demonstrated that LPC could cause a rapid, dose-dependent increase in the [Ca(2+)]i in the presence of external calcium ([Ca(2+)]e). ... The LPC-induced [Ca(2+)]i increase in S2 cells characterized in this study may shed light on the study of NTE/SWS protein function in general because the enzyme is responsible for the deacylation of LPC."
Friday, July 17, 2015
in vivo RNAi contributes to follow-up of GWAS candidate aggression-related genes
Shorter J, Couch C, Huang W, Carbone MA, Peiffer J, Anholt RR, Mackay TF. Genetic architecture of natural variation in Drosophila melanogaster aggressive behavior. Proc Natl Acad Sci U S A. 2015 Jul 7;112(27):E3555-63. PMID: 26100892.
From the abstract: "Aggression is an evolutionarily conserved complex behavior ... Here, we performed genome-wide association analyses using the inbred, sequenced lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and replicate advanced intercross populations derived from the most and least aggressive DGRP lines. We identified genes that have been previously implicated in aggressive behavior as well as many novel loci ... We used mutations and RNAi knock-down alleles to functionally validate 79% of the candidate genes and 75% of the candidate epistatic interactions tested. Epistasis for aggressive behavior causes cryptic genetic variation in the DGRP that is revealed by changing allele frequencies in the outbred populations derived from extreme DGRP lines. This phenomenon may pertain to other fitness traits and species, with implications for evolution, applied breeding, and human genetics."
From the abstract: "Aggression is an evolutionarily conserved complex behavior ... Here, we performed genome-wide association analyses using the inbred, sequenced lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and replicate advanced intercross populations derived from the most and least aggressive DGRP lines. We identified genes that have been previously implicated in aggressive behavior as well as many novel loci ... We used mutations and RNAi knock-down alleles to functionally validate 79% of the candidate genes and 75% of the candidate epistatic interactions tested. Epistasis for aggressive behavior causes cryptic genetic variation in the DGRP that is revealed by changing allele frequencies in the outbred populations derived from extreme DGRP lines. This phenomenon may pertain to other fitness traits and species, with implications for evolution, applied breeding, and human genetics."
Report explores mechanisms underlying target cleavage by RISC
Yao C, Sasaki HM, Ueda T, Tomari Y, Tadakuma H. Single-Molecule Analysis of the Target Cleavage Reaction by the Drosophila RNAi Enzyme Complex. Mol Cell. 2015 Jul 2;59(1):125-32. PMID:
26140368.
From the abstract: "Small interfering RNAs (siRNAs) direct cleavage of complementary target RNAs via an RNA-induced silencing complex (RISC) that contains Argonatute2 protein at its core. However, what happens after target cleavage remains unclear. Here we analyzed the cleavage reaction by Drosophila Argonaute2-RISC using single-molecule imaging and revealed a series of intermediate states in target recognition, cleavage, and product release. Our data suggest that, after cleavage, RISC generally releases the 5' cleavage fragment from the guide 3' supplementary region first and then the 3' fragment from the seed region, highlighting the reinforcement of the seed pairing in RISC. ... "
From the abstract: "Small interfering RNAs (siRNAs) direct cleavage of complementary target RNAs via an RNA-induced silencing complex (RISC) that contains Argonatute2 protein at its core. However, what happens after target cleavage remains unclear. Here we analyzed the cleavage reaction by Drosophila Argonaute2-RISC using single-molecule imaging and revealed a series of intermediate states in target recognition, cleavage, and product release. Our data suggest that, after cleavage, RISC generally releases the 5' cleavage fragment from the guide 3' supplementary region first and then the 3' fragment from the seed region, highlighting the reinforcement of the seed pairing in RISC. ... "
Fly cell screen leads to findings relevant to cancer treatment (and its financial impact)
Thomas S, Fisher KH, Snowden JA, Danson SJ, Brown S, Zeidler MP. Methotrexate Is a JAK/STAT Pathway Inhibitor. PLoS One. 2015 Jul 1;10(7):e0130078. PMID: 26131691.
From the abstract: "... The JAK/STAT pathway transduces signals from multiple cytokines and controls haematopoiesis, immunity and inflammation. In addition, pathological activation is seen in multiple malignancies including the myeloproliferative neoplasms (MPNs). Given this, drug development efforts have targeted the pathway with JAK inhibitors such as ruxolitinib. Although effective, high costs and side effects have limited its adoption. Thus, a need for effective low cost treatments remains. ...We used the low-complexity Drosophila melanogaster pathway to screen for small molecules that modulate JAK/STAT signalling. This screen identified methotrexate and the closely related aminopterin as potent suppressors of STAT activation. We show that methotrexate suppresses human JAK/STAT signalling without affecting other phosphorylation-dependent pathways. ... While the JAK1/2 inhibitor ruxolitinib is effective, a £43,200 annual cost precludes widespread adoption. With an annual methotrexate cost of around £32, our findings represent an important development with significant future potential."
From the abstract: "... The JAK/STAT pathway transduces signals from multiple cytokines and controls haematopoiesis, immunity and inflammation. In addition, pathological activation is seen in multiple malignancies including the myeloproliferative neoplasms (MPNs). Given this, drug development efforts have targeted the pathway with JAK inhibitors such as ruxolitinib. Although effective, high costs and side effects have limited its adoption. Thus, a need for effective low cost treatments remains. ...We used the low-complexity Drosophila melanogaster pathway to screen for small molecules that modulate JAK/STAT signalling. This screen identified methotrexate and the closely related aminopterin as potent suppressors of STAT activation. We show that methotrexate suppresses human JAK/STAT signalling without affecting other phosphorylation-dependent pathways. ... While the JAK1/2 inhibitor ruxolitinib is effective, a £43,200 annual cost precludes widespread adoption. With an annual methotrexate cost of around £32, our findings represent an important development with significant future potential."
Protocol paper -- qPCR to detect cell death-related transcripts following dsRNA treatment of fly cells
Denton D, Kumar S. Analyzing the Response of RNAi-Treated Drosophila Cells to Death Stimuli by Quantitative Real-Time Polymerase Chain Reaction. Cold Spring Harb Protoc. 2015 Jul 1;2015(7):pdb.prot086223. PMID: 26134907.
From the abstract: "A useful complement to animal studies is the use of Drosophila cell lines to analyze cell-death responses. ... Drosophila cell lines are very amenable to knockdown studies ... the cell lines are useful for investigating the response to death stimuli, following gene knockdown, by examining the expression of cell-death genes. This protocol describes the synthesis of dsRNA for treatment of Drosophila cells and the subsequent analysis of cell-death gene expression by quantitative real-time polymerase chain reaction (qPCR)."
From the abstract: "A useful complement to animal studies is the use of Drosophila cell lines to analyze cell-death responses. ... Drosophila cell lines are very amenable to knockdown studies ... the cell lines are useful for investigating the response to death stimuli, following gene knockdown, by examining the expression of cell-death genes. This protocol describes the synthesis of dsRNA for treatment of Drosophila cells and the subsequent analysis of cell-death gene expression by quantitative real-time polymerase chain reaction (qPCR)."
Thursday, July 16, 2015
Methods paper--RNAi screening and the centrosome
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: "... Genome-wide RNA interference (RNAi) allows for comprehensive screening ... Drosophila tissue culture cells provide an attractive model for such screens. ... Drosophila centrosomes are similar to their human counterparts, but less complex. Thus, all major centrosome components are conserved and fewer redundancies apply ... 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."
From the abstract: "... Genome-wide RNA interference (RNAi) allows for comprehensive screening ... Drosophila tissue culture cells provide an attractive model for such screens. ... Drosophila centrosomes are similar to their human counterparts, but less complex. Thus, all major centrosome components are conserved and fewer redundancies apply ... 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."
Thursday, July 9, 2015
Fly gene list resource live and published
The DRSC is pleased to say that our GLAD gene list annotation database is live at flyrnai.org/glad and published in the Journal of Genomics!
The tool lets you view curated gene lists, corresponding information, and more. Improvable through community input on genes to add, publications to check out, etc. relevant to specific litst.
The tool lets you view curated gene lists, corresponding information, and more. Improvable through community input on genes to add, publications to check out, etc. relevant to specific litst.
Wednesday, June 24, 2015
Search with "Drosophila" now returns >90,000 results in PubMed!
#Drosophila now returns >90,000 results at PubMed. Let the count down to >100,000 begin!!! — Stephanie Mohr (@smohrfly) June 24, 2015
"Genetic toolkit" for intronic MiMICs
Nagarkar-Jaiswal S, DeLuca SZ, Lee PT, Lin WW, Pan H, Zuo Z, Lv J, Spradling AC, Bellen HJ. A genetic toolkit for tagging intronic MiMIC containing genes. Elife. 2015 Jun 23;4. PMID: 26102525.
From the abstract: "... Here we describe a simple and reliable genetic strategy to tag genes/proteins that contain MiMIC insertions using an integrated exon encoding GFP flanked by FRT sequences. We document the efficiency and tag 60 mostly uncharacterized genes."
Study uses transcriptomics and in vivo RNAi follow-up to explore sleep
Tomita J, Ueno T, Mitsuyoshi M, Kume S, Kume K. The NMDA Receptor Promotes Sleep in the Fruit Fly, Drosophila melanogaster. PLoS One. 2015 May 29;10(5):e0128101. PMID: 26023770; PMCID: PMC4449117.
From the abstract: "... sleep is essential for learning and memory. Drosophila melanogaster has emerged as a novel model for studying sleep. ... we carried out a microarray analysis comparing mRNA expression in heads of fmn and control flies and found that 563 genes are differentially expressed. Next, using the pan-neuronal Gal4 driver elav-Gal4 and UAS-RNA interference (RNAi) to knockdown individual genes, we performed a functional screen. We found that knockdown of the NMDA type glutamate receptor channel gene (Nmdar1) (also known as dNR1) reduced sleep. ..."
From the abstract: "... sleep is essential for learning and memory. Drosophila melanogaster has emerged as a novel model for studying sleep. ... we carried out a microarray analysis comparing mRNA expression in heads of fmn and control flies and found that 563 genes are differentially expressed. Next, using the pan-neuronal Gal4 driver elav-Gal4 and UAS-RNA interference (RNAi) to knockdown individual genes, we performed a functional screen. We found that knockdown of the NMDA type glutamate receptor channel gene (Nmdar1) (also known as dNR1) reduced sleep. ..."
Caffeine, kinases and the Hippo pathway: cell-based fly RNAi screen with in vivo follow-up
Di Cara F, Maile TM, Parsons BD, Magico A, Basu S, Tapon N, King-Jones K. The Hippo pathway promotes cell survival in response to chemical stress. Cell Death Differ. 2015 Mar 13. PMID: 26021298.
From the abstract: "Cellular stress defense mechanisms have evolved to maintain homeostasis in response to a broad variety of environmental challenges. Stress signaling pathways activate multiple cellular programs that range from the activation of survival pathways to the initiation of cell death when cells are damaged beyond repair. To identify novel players acting in stress response pathways, we conducted a cell culture RNA interference (RNAi) screen using caffeine as a xenobiotic stress-inducing agent, as this compound is a well-established inducer of detoxification response pathways. Specifically, we examined how caffeine affects cell survival when Drosophila kinases and phosphatases were depleted via RNAi. Using this approach, we identified and validated 10 kinases and 4 phosphatases that are essential for cell survival under caffeine-induced stress both in cell culture and living flies. Remarkably, our screen yielded an enrichment of Hippo pathway components, indicating that this pathway regulates cellular stress responses. ... Our in vitro and in vivo loss-of-function data therefore implicate Hippo signaling in the transduction of cellular survival signals in response to chemical stress."
From the abstract: "Cellular stress defense mechanisms have evolved to maintain homeostasis in response to a broad variety of environmental challenges. Stress signaling pathways activate multiple cellular programs that range from the activation of survival pathways to the initiation of cell death when cells are damaged beyond repair. To identify novel players acting in stress response pathways, we conducted a cell culture RNA interference (RNAi) screen using caffeine as a xenobiotic stress-inducing agent, as this compound is a well-established inducer of detoxification response pathways. Specifically, we examined how caffeine affects cell survival when Drosophila kinases and phosphatases were depleted via RNAi. Using this approach, we identified and validated 10 kinases and 4 phosphatases that are essential for cell survival under caffeine-induced stress both in cell culture and living flies. Remarkably, our screen yielded an enrichment of Hippo pathway components, indicating that this pathway regulates cellular stress responses. ... Our in vitro and in vivo loss-of-function data therefore implicate Hippo signaling in the transduction of cellular survival signals in response to chemical stress."
Thursday, June 18, 2015
miRNA "sponge" fly stock resource
Fulga TA, McNeill EM, Binari R, Yelick J, Blanche A, Booker M, Steinkraus BR, Schnall-Levin M, Zhao Y, DeLuca T, Bejarano F, Han Z, Lai EC, Wall DP, Perrimon N, Van Vactor D. A transgenic resource for conditional competitive inhibition of conserved Drosophila microRNAs. Nat Commun. 2015 Jun 17;6:7279. PMID: 26081261.
From the abstract: "... Development of competitive inhibitor molecules such as miRNA sponges has allowed the community to address individual miRNA function in vivo. ... Here we offer a comprehensive library of 141 conditional miRNA sponges targeting well-conserved miRNAs in Drosophila. ..."
From the abstract: "... Development of competitive inhibitor molecules such as miRNA sponges has allowed the community to address individual miRNA function in vivo. ... Here we offer a comprehensive library of 141 conditional miRNA sponges targeting well-conserved miRNAs in Drosophila. ..."
Wednesday, June 17, 2015
Sneak peak for blog readers: Gene List Annotation for Drosophila (GLAD) at DRSC
For years the DRSC has been putting together gene lists, e.g. to build focused cell-based RNAi libraries like our kinases and phosphatases library.
We recently built an online tool, GLAD, to support search and view of the several gene lists put together by Hu et al. over the years. When possible, the lists are annotated with information regarding confidence. Some lists are based on biochemical function (e.g. kinases) and others are based on biological function (e.g. autophagy). Check it out!
A companion paper has been accepted by Journal of Genomics. I'll post when it's out.
At the online tool, you can suggest additions or changes to the list, or suggest relevant papers, so that we can improve the resource over time.
We have also made it possible for the community to quickly use a gene list to find relevant TRiP and other RNAi fly stocks in public collections.
See also related post on the complementary gene groups resource at FlyBase.
We recently built an online tool, GLAD, to support search and view of the several gene lists put together by Hu et al. over the years. When possible, the lists are annotated with information regarding confidence. Some lists are based on biochemical function (e.g. kinases) and others are based on biological function (e.g. autophagy). Check it out! A companion paper has been accepted by Journal of Genomics. I'll post when it's out.
At the online tool, you can suggest additions or changes to the list, or suggest relevant papers, so that we can improve the resource over time.
We have also made it possible for the community to quickly use a gene list to find relevant TRiP and other RNAi fly stocks in public collections.
See also related post on the complementary gene groups resource at FlyBase.
C. elegans study points to pol theta involvement in CRISPR mediated events
From our worm colleagues--article of possible interest to folks working with CRISPR system:
van Schendel R, Roerink SF, Portegijs V, van den Heuvel S, Tijsterman M. Polymerase Θ is a key driver of genome evolution and of CRISPR/Cas9-mediated mutagenesis. Nat Commun. 2015 Jun 16;6:7394. PMID: 26077599.
From the abstract: "... Here, we demonstrate that the A-family polymerase theta (POLQ) is a major driver of inheritable genomic alterations in Caenorhabditis elegans. Unlike somatic cells, which use non-homologous end joining (NHEJ) to repair DNA transposon-induced DSBs, germ cells use polymerase theta-mediated end joining, a conceptually simple repair mechanism requiring only one nucleotide as a template for repair. Also CRISPR/Cas9-induced genomic changes are exclusively generated through polymerase theta-mediated end joining, refuting a previously assumed requirement for NHEJ in their formation. ... "
van Schendel R, Roerink SF, Portegijs V, van den Heuvel S, Tijsterman M. Polymerase Θ is a key driver of genome evolution and of CRISPR/Cas9-mediated mutagenesis. Nat Commun. 2015 Jun 16;6:7394. PMID: 26077599.
From the abstract: "... Here, we demonstrate that the A-family polymerase theta (POLQ) is a major driver of inheritable genomic alterations in Caenorhabditis elegans. Unlike somatic cells, which use non-homologous end joining (NHEJ) to repair DNA transposon-induced DSBs, germ cells use polymerase theta-mediated end joining, a conceptually simple repair mechanism requiring only one nucleotide as a template for repair. Also CRISPR/Cas9-induced genomic changes are exclusively generated through polymerase theta-mediated end joining, refuting a previously assumed requirement for NHEJ in their formation. ... "
Tuesday, June 16, 2015
New online resource for TALE design: SIFTED
The Bulyk lab at Brigham and Women's Hospital has launched the software suite SIFTED, which should be useful to folks designing or interpreting results using Tal Effector Nucleases (TALEs).
Corresponding publication: Rogers JM, Barrera LA, Reyon D, Sander JD, Kellis M, Keith Joung J, Bulyk ML. Context influences on TALE-DNA binding revealed by quantitative profiling. Nat Commun. 2015 Jun 11;6:7440. PMID: 26067805.
Corresponding publication: Rogers JM, Barrera LA, Reyon D, Sander JD, Kellis M, Keith Joung J, Bulyk ML. Context influences on TALE-DNA binding revealed by quantitative profiling. Nat Commun. 2015 Jun 11;6:7440. PMID: 26067805.
Tuesday, June 2, 2015
Actin in the nucleus -- genome-wide cell-based RNAi screen
Excited to see this report on a screen performed by J. Dopie at the DRSC!
Dopie J, Rajakylä EK, Joensuu MS, Huet G, Ferrantelli E, Xie T, Jäälinoja H, Jokitalo E, Vartiainen MK. Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators. J Cell Sci. 2015 May 28. pii: jcs.169441. PMID: 26021350.
From the abstract: "Nuclear actin plays an important role in many processes that regulate gene expression. ... Here we have performed a genome-wide RNAi screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo/Bach2, SNF4Aγ/Prkag1 and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several novel regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. ... Our screen therefore reveals novel aspects of actin regulation and links nuclear actin to many cellular processes."
Dopie J, Rajakylä EK, Joensuu MS, Huet G, Ferrantelli E, Xie T, Jäälinoja H, Jokitalo E, Vartiainen MK. Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators. J Cell Sci. 2015 May 28. pii: jcs.169441. PMID: 26021350.
From the abstract: "Nuclear actin plays an important role in many processes that regulate gene expression. ... Here we have performed a genome-wide RNAi screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo/Bach2, SNF4Aγ/Prkag1 and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several novel regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. ... Our screen therefore reveals novel aspects of actin regulation and links nuclear actin to many cellular processes."
Wednesday, May 27, 2015
in vivo RNAi screen for "flyabetes" phenotype reveals insight into mammalian systems
Ugrankar R, Berglund E, Akdemir F, Tran C, Kim MS, Noh J, Schneider R, Ebert B, Graff JM. Drosophila glucome screening identifies Ck1alpha as a regulator of mammalian glucose metabolism. Nat Commun. 2015 May 21;6:7102. PMID: 25994086.
Tuesday, May 26, 2015
IMP integrative tool for functional genomics updated to 2.0 version
Wong AK, Krishnan A, Yao V, Tadych A, Troyanskaya OG. IMP 2.0: a multi-species functional genomics portal for integration, visualization and prediction of protein functions and networks. Nucleic Acids Res. 2015 May 12. PMID: 25969450.
From the abstract: "... IMP 2.0 integrates updated prior knowledge and data collections from the last three years in the seven supported organisms (Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Danio rerio, Caenorhabditis elegans, and Saccharomyces cerevisiae) and extends function prediction coverage to include human disease. ... Additionally, IMP 2.0 implements a new flexible platform for experts to generate custom hypotheses about biological processes or diseases, making sophisticated data-driven methods easily accessible to researchers. IMP does not require any registration or installation and is freely available for use at http://imp.princeton.edu."
From the abstract: "... IMP 2.0 integrates updated prior knowledge and data collections from the last three years in the seven supported organisms (Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Danio rerio, Caenorhabditis elegans, and Saccharomyces cerevisiae) and extends function prediction coverage to include human disease. ... Additionally, IMP 2.0 implements a new flexible platform for experts to generate custom hypotheses about biological processes or diseases, making sophisticated data-driven methods easily accessible to researchers. IMP does not require any registration or installation and is freely available for use at http://imp.princeton.edu."
Friday, May 22, 2015
Registration and abstract submission open for 24th European Drosophila Research Conference
Registration and abstract submission are open for 24th European Drosophila Research Conference, which will be held September 9-12, 2015 in Heidelberg, Germany.
Thursday, May 21, 2015
CRISPRs in flies -- two papers
Protocol recently published:
Gratz SJ, Harrison MM, Wildonger J, O'Connor-Giles KM. Precise Genome Editing of Drosophila with CRISPR RNA-Guided Cas9. Methods Mol Biol. 2015;1311:335-48. PMID: 25981484.
And early online at G3:
Systematic Evaluation of Drosophila CRISPR Tools Reveals Safe and Robust Alternatives to Autonomous Gene Drives in Basic Research
Gratz SJ, Harrison MM, Wildonger J, O'Connor-Giles KM. Precise Genome Editing of Drosophila with CRISPR RNA-Guided Cas9. Methods Mol Biol. 2015;1311:335-48. PMID: 25981484.
And early online at G3:
Systematic Evaluation of Drosophila CRISPR Tools Reveals Safe and Robust Alternatives to Autonomous Gene Drives in Basic Research
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Wednesday, May 13, 2015
Using phylogenetic relationships to predict protein functions--Sadreyev et al. release PhyloGene
In the 'not RNAi (or CRISPR) but might be of interest to blog readers anyway' department:
Sadreyev IR, Ji F, Cohen E, Ruvkun G, Tabach Y. PhyloGene server for identification and visualization of co-evolving proteins using normalized phylogenetic profiles. Nucleic Acids Res. 2015 May 9. pii: gkv452. PMID: 25958392.
From the abstract: "... Analysis of the similarity in patterns of sequence conservation across a large set of eukaryotes can predict functional associations between different proteins, identify new pathway members and reveal the function of previously uncharacterized proteins. We used normalized phylogenetic profiling to predict protein function and identify new pathway members and disease genes. The phylogenetic profiles of tens of thousands conserved proteins in the human, mouse, Caenorhabditis elegans and Drosophila genomes can be queried on our new web server, PhyloGene. ..."
Sadreyev IR, Ji F, Cohen E, Ruvkun G, Tabach Y. PhyloGene server for identification and visualization of co-evolving proteins using normalized phylogenetic profiles. Nucleic Acids Res. 2015 May 9. pii: gkv452. PMID: 25958392.
From the abstract: "... Analysis of the similarity in patterns of sequence conservation across a large set of eukaryotes can predict functional associations between different proteins, identify new pathway members and reveal the function of previously uncharacterized proteins. We used normalized phylogenetic profiling to predict protein function and identify new pathway members and disease genes. The phylogenetic profiles of tens of thousands conserved proteins in the human, mouse, Caenorhabditis elegans and Drosophila genomes can be queried on our new web server, PhyloGene. ..."
Monday, May 4, 2015
FlyBase announces "Gene Group Reports"
FlyBase's May release includes "Gene Group Reports" (see FlyBase release notes news). I was able to view a list of all gene groups by entering the wild card "*" at the Simple tab on the Quick Search link box on the FlyBase home page.
GenomeRNAi announces data release v.14
The DKFZ's GenomeRNAi recently announced that "The database now contains phenotype data from 217 human RNAi screens and 201 screens in D. melanogaster. The total number of gene-phenotype associations has increased to over 1,2 million." That's a lot of associations!
Friday, April 10, 2015
Transcription factor validated in follow-up to genome-wide RNAi screen related to viral-host cell interactions
Panda D, Gold B, Tartell MA, Rausch K, Casas-Tinto S, Cherry S. The Transcription Factor FoxK Participates with Nup98 To Regulate Antiviral Gene Expression. MBio. 2015 Apr 7;6(2). PMID: 25852164.
in vivo screen of kinases and phosphatases identifies Wnt signal regulators
Swarup S, Pradhan-Sundd T, Verheyen EM. Genome-wide identification of phospho-regulators of Wnt signaling in Drosophila. Development. 2015 Apr 15;142(8):1502-15. PMID: 25852200.
From the abstract: "... In this study, we performed a genome-wide in vivo RNAi screen for kinases and phosphatases that regulate the Wnt pathway under physiological conditions in the Drosophila wing disc. Our analyses have identified 54 high-confidence kinases and phosphatases capable of modulating the Wnt pathway, including 22 novel regulators. These candidates were also assayed for a role in the Notch pathway ... Additionally, each regulator of the Wnt pathway was evaluated in the wing disc for its ability to affect the mechanistically similar Hedgehog pathway. ... "
From the abstract: "... In this study, we performed a genome-wide in vivo RNAi screen for kinases and phosphatases that regulate the Wnt pathway under physiological conditions in the Drosophila wing disc. Our analyses have identified 54 high-confidence kinases and phosphatases capable of modulating the Wnt pathway, including 22 novel regulators. These candidates were also assayed for a role in the Notch pathway ... Additionally, each regulator of the Wnt pathway was evaluated in the wing disc for its ability to affect the mechanistically similar Hedgehog pathway. ... "
Wednesday, April 8, 2015
Report of improved CRISPR-based transcriptional activation approach includes work in Drosophila cultured cells
Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R Iyer E, Lin S, Kiani S, Guzman CD, Wiegand DJ, Ter-Ovanesyan D, Braff JL, Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM. Highly efficient Cas9-mediated transcriptional programming. Nat Methods. 2015 Apr;12(4):326-8. PMID: 25730490.
From the abstract: "... modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. ..."
Drosophila S2R+ cultured cells are among the systems in which this approach was successfully tested. See Results and Supplementary Fig. 9.
From the abstract: "... modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. ..."
Drosophila S2R+ cultured cells are among the systems in which this approach was successfully tested. See Results and Supplementary Fig. 9.
Drosophila Models of Human Disease: Mammalian screen with fly follow-up identifies pot...
Drosophila Models of Human Disease: Mammalian screen with fly follow-up identifies pot...: Jimenez-Sanchez M, Lam W, Hannus M, Sönnichsen B, Imarisio S, Fleming A, Tarditi A, Menzies F, Ed Dami T, Xu C, Gonzalez-Couto E, Lazzeroni ...
C. elegans report presents simple rule for effective CRISPR sgRNA design
Farboud B, Meyer BJ. Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design. Genetics. 2015 Feb 18. PMID: 25695951.
From the abstract: "... Here we describe and validate a strategy for Caenorhabditis elegans ... The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. ..."
Will this prove true in Drosophila as well? There are certainly guide designs reported to be effective in flies that break the GG rule. And it is hard to believe that the design algorithms developed by various groups would not have picked this up. Nevertheless, it's tempting to give it a try.
From the abstract: "... Here we describe and validate a strategy for Caenorhabditis elegans ... The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. ..."
Will this prove true in Drosophila as well? There are certainly guide designs reported to be effective in flies that break the GG rule. And it is hard to believe that the design algorithms developed by various groups would not have picked this up. Nevertheless, it's tempting to give it a try.
Monday, April 6, 2015
Efficient fly CRISPR with limited risk? Preprint from Port and colleagues compares sgRNA transgenic and MCR approaches
Interested to efficiently generate knockout flies without the potential risks presented by the mutagenic chain reaction (MCR) approach? Check out a new pre-print from Port et al.
Port, Muschalik and Bullock (preprint) CRISPR with independent transgenes is a safe and robust alternative to autonomous gene drives in basic research.
From the abstract: "... Here, we systematically evaluate available CRISPR/Cas reagents and experimental designs in Drosophila. Our findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. We perform gene editing at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. We contrast our method to recently developed autonomous gene drive technology for genome engineering (Gantz & Bier, 2015) and conclude that optimised CRISPR with independent transgenes is as efficient, more versatile and does not represent a biosafety risk."
Port, Muschalik and Bullock (preprint) CRISPR with independent transgenes is a safe and robust alternative to autonomous gene drives in basic research.
From the abstract: "... Here, we systematically evaluate available CRISPR/Cas reagents and experimental designs in Drosophila. Our findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. We perform gene editing at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. We contrast our method to recently developed autonomous gene drive technology for genome engineering (Gantz & Bier, 2015) and conclude that optimised CRISPR with independent transgenes is as efficient, more versatile and does not represent a biosafety risk."
Monday, March 30, 2015
NIH NIGMS announces new strategic plan
Many readers will be interested to learn that NIGMS at NIH has released a new strategic plan. An announcement, including a link to a PDF copy of the plan, is at the NIGMS Feedback Loop blog.
Report of 'systems-level interrogation' includes DRSC RNAi screen data, phosphoproteomics and in vivo analyses
Sopko R, Lin YB, Makhijani K, Alexander B, Perrimon N, Brückner K. A systems-level interrogation identifies regulators of Drosophila blood cell number and survival. PLoS Genet. 2015 Mar 6;11(3):e1005056. PMID: 25749252; PMCID: PMC4352040.
From the abstract: "Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. ... Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and ... Ecdysone receptor (EcR) and ultraspiracle (usp) ... as Pvr Suppressors. ... Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. ... our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems."
From the abstract: "Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. ... Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and ... Ecdysone receptor (EcR) and ultraspiracle (usp) ... as Pvr Suppressors. ... Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. ... our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems."
Friday, March 27, 2015
GenomeRNAi beta-testing a 'screen comparison tool'
I recently stumbled across this beta-test at GenomeRNAi of a screen comparison tool. Enter one or a list of genes and view, heat-map style, in what screens those genes were hits. They indicate they're interested to receive feedback. The default option is human genes but Drosophila also supported.
Wednesday, March 25, 2015
"Trojan exon" variation on the theme of MiMIC and CRISPR insertion strategies
Diao F, Ironfield H, Luan H, Diao F, Shropshire WC, Ewer J, Marr E, Potter CJ, Landgraf M, White BH. Plug-and-Play Genetic Access to Drosophila Cell Types using Exchangeable Exon Cassettes. Cell Rep. 2015 Mar 3;10(8):1410-21. PMID: 25732830.
From the abstract: "... we introduce a simple, versatile method for achieving cell-type-specific expression of transgenes ... Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest ... We demonstrate the efficacy of this approach in Drosophila ... We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system."
From the abstract: "... we introduce a simple, versatile method for achieving cell-type-specific expression of transgenes ... Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest ... We demonstrate the efficacy of this approach in Drosophila ... We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system."
Protocol report--dual luciferase assays
Yun C, Dasgupta R. Luciferase reporter assay in Drosophila and mammalian tissue culture cells. Curr Protoc Chem Biol. 2014 Mar 14;6(1):7-23. PMID: 24652620; PMCID: PMC4059354.
Friday, March 20, 2015
The cut that keeps on cutting: CRISPR-Cas mutagenic chain reaction approach
Gantz and Bier (2015) The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations. Science early online DOI: 10.1126/science.aaa5945
Abstract: "An organism with a single recessive loss-of-function allele will typically have a wild-type phenotype while individuals homozygous for two copies of the allele will display a mutant phenotype. Here, we develop a method that we refer to as the mutagenic chain reaction (MCR), which is based on the CRISPR/Cas9 genome editing system for generating autocatalytic mutations to generate homozygous loss-of-function mutations. We demonstrate in Drosophila that MCR mutations efficiently spread from their chromosome of origin to the homologous chromosome thereby converting heterozygous mutations to homozygosity in the vast majority of somatic and germline cells. MCR technology should have broad applications in diverse organisms."
See also Science magazine's news coverage of the report.
Abstract: "An organism with a single recessive loss-of-function allele will typically have a wild-type phenotype while individuals homozygous for two copies of the allele will display a mutant phenotype. Here, we develop a method that we refer to as the mutagenic chain reaction (MCR), which is based on the CRISPR/Cas9 genome editing system for generating autocatalytic mutations to generate homozygous loss-of-function mutations. We demonstrate in Drosophila that MCR mutations efficiently spread from their chromosome of origin to the homologous chromosome thereby converting heterozygous mutations to homozygosity in the vast majority of somatic and germline cells. MCR technology should have broad applications in diverse organisms."
See also Science magazine's news coverage of the report.
Wednesday, March 18, 2015
CRISPR off-targets study in human cells
Findings relevant to fly studies? Will be interesting to see.
Kim D, Bae S, Park J, Kim E, Kim S, Yu HR, Hwang J, Kim JI, Kim JS. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015 Mar;12(3):237-43. PMID: 25664545.
From the abstract: "... genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. ... We also showed that Cas9 nucleases can be highly specific ... and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. ..."
Kim D, Bae S, Park J, Kim E, Kim S, Yu HR, Hwang J, Kim JI, Kim JS. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015 Mar;12(3):237-43. PMID: 25664545.
From the abstract: "... genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. ... We also showed that Cas9 nucleases can be highly specific ... and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. ..."
Tuesday, March 17, 2015
Review of fly CRISPR approaches
Jiang Xua, Xingjie Rena, Jin Suna, Xia Wanga, Huan-Huan Qiaoa, Bo-Wen Xua, Lu-Ping Liua, Jian-Quan Ni (2014) A Toolkit of CRISPR-Based Genome Editing Systems in Drosophila. In press manuscript available at Journal of Genetics and Genomics
From the abstract: "... we focus on the applications of this popular system in Drosophila. We discuss the effectiveness of the CRISPR/Cas9 systems in terms of delivery, mutagenesis detection, parameters affecting efficiency, and off-target issues, with an emphasis on how to apply this powerful tool to characterize gene functions."
See also: igtrcn.org/pulling-recent-crisprcas9-work-in-drosophila-together/
From the abstract: "... we focus on the applications of this popular system in Drosophila. We discuss the effectiveness of the CRISPR/Cas9 systems in terms of delivery, mutagenesis detection, parameters affecting efficiency, and off-target issues, with an emphasis on how to apply this powerful tool to characterize gene functions."
See also: igtrcn.org/pulling-recent-crisprcas9-work-in-drosophila-together/
Wednesday, March 4, 2015
Fly person? Get yourself counted!
The Genetics Society of America and other groups advocating for Drosophila research and research funding can benefit from learning up-to-date numbers of Drosophila researchers. How can you help? Get counted. Follow this link to self-identify as a fly person at FlyBase.
Thursday, February 26, 2015
New Gal4 drivers for follicle cell studies
Hartman TR, Ventresca EM, Hopkins A, Zinshteyn D, Singh T, O'Brien JA, Neubert BC, Hartman MG, Schofield HK, Stavrides KP, Talbot DE, Riggs DJ, Pritchard C, O'Reilly AM. Novel Tools for Genetic Manipulation of Follicle Stem Cells in the Drosophila Ovary Reveal an Integrin-Dependent Transition from Quiescence to Proliferation. Genetics. 2015 Feb 12. PMID: 25680813.
From the abstract: "... Here, we present novel methods for marking and genetically altering epithelial Follicle Stem Cells (FSCs) within the Drosophila ovary. ..."
From the abstract: "... Here, we present novel methods for marking and genetically altering epithelial Follicle Stem Cells (FSCs) within the Drosophila ovary. ..."
Tuesday, February 24, 2015
Primary cell screen performed at DRSC explores autophagy in muscles--relevance to glycogen storage disorders
Zirin J, Nieuwenhuis J, Samsonova A, Tao R, Perrimon N. Regulators of autophagosome formation in Drosophila muscles. PLoS Genet. 2015 Feb 18;11(2):e1005006. PMID: 25692684.
From the abstract: "... We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells ... The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. ... In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function."
The DRSC autophagy library used in the screen is one of several focused libraries available from the Drosophila RNAi Screening Center for on-site or off-site screens.
From the abstract: "... We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells ... The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. ... In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function."
The DRSC autophagy library used in the screen is one of several focused libraries available from the Drosophila RNAi Screening Center for on-site or off-site screens.
Genome-wide in vivo RNAi and data integration featured in study on intestinal stem cell regulation
Zeng X, Han L, Singh SR, Liu H, Neumüller RA, Yan D, Hu Y, Liu Y, Liu W, Lin X, Hou SX. Genome-wide RNAi Screen Identifies Networks Involved in Intestinal Stem Cell Regulation in Drosophila. Cell Rep. 2015 Feb 17. pii: S2211-1247(15)00076-5. PMID: 25704823.
From the abstract: "... To comprehensively identify genes and pathways that regulate ISC fates, we performed a genome-wide transgenic RNAi screen in adult Drosophila intestine and identified 405 genes that regulate ISC maintenance and lineage-specific differentiation. By integrating these genes into publicly available interaction databases, we further developed functional networks that regulate ..."
From the abstract: "... To comprehensively identify genes and pathways that regulate ISC fates, we performed a genome-wide transgenic RNAi screen in adult Drosophila intestine and identified 405 genes that regulate ISC maintenance and lineage-specific differentiation. By integrating these genes into publicly available interaction databases, we further developed functional networks that regulate ..."
Monday, February 23, 2015
Review of model organism RNAi screen contributions to understanding host-pathogen interactions
Abnave P, Conti F, Torre C, Ghigo E. What RNAi screens in model organisms revealed about microbicidal response in mammals? Front Cell Infect Microbiol.
2015 Jan 12;4:184. PMID: 25629007; PMCID: PMC4290690.
From the abstract: "The strategies evolved by pathogens to infect hosts and the mechanisms used by the host to eliminate intruders are highly complex. ... The aim of this mini-review is to highlight and describe recent breakthroughs in the field of host-pathogen interactions using RNAi screens of model organisms. We will focus specifically on the model organisms Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio. Moreover, a recent study examining the immune system of planarian will be discussed."
From the abstract: "The strategies evolved by pathogens to infect hosts and the mechanisms used by the host to eliminate intruders are highly complex. ... The aim of this mini-review is to highlight and describe recent breakthroughs in the field of host-pathogen interactions using RNAi screens of model organisms. We will focus specifically on the model organisms Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio. Moreover, a recent study examining the immune system of planarian will be discussed."
Sleep screens--methods review includes discussion of in vivo RNAi
Axelrod S, Saez L, Young MW. Studying circadian rhythm and sleep using genetic screens in Drosophila. Methods Enzymol. 2015;551:3-27. PMID: 25662449.
From the abstract: "... In this chapter, we briefly recall the history of circadian rhythm and sleep screens and then ... describe techniques ... for ... screening in the field. ... comparing the newer approaches of transgenic RNA interference (RNAi) to classical forms of mutagenesis ... We discuss the different screening approaches in light of the literature and published and unpublished sleep and rhythm screens utilizing ethyl methanesulfonate mutagenesis and transgenic RNAi from our lab."
From the abstract: "... In this chapter, we briefly recall the history of circadian rhythm and sleep screens and then ... describe techniques ... for ... screening in the field. ... comparing the newer approaches of transgenic RNA interference (RNAi) to classical forms of mutagenesis ... We discuss the different screening approaches in light of the literature and published and unpublished sleep and rhythm screens utilizing ethyl methanesulfonate mutagenesis and transgenic RNAi from our lab."
Friday, February 13, 2015
DRSC website re-vamp coming soon
Here's a view of the revised DRSC home page, soon to be launched at www.flyrnai.org
Our primary goals were to facilitate improved navigation of online tools and to improve compatibility with hand-held devices. Feedback is welcome, as always!
Our primary goals were to facilitate improved navigation of online tools and to improve compatibility with hand-held devices. Feedback is welcome, as always!
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| close-up of the research workflow-based view of our online tools. text = links (on the real version--not here!) |
Thursday, February 5, 2015
Fat body RNAi screen identifies matrix metalloproteins
Jia Q, Liu Y, Liu H, Li S. Mmp1 and Mmp2 cooperatively induce Drosophila fat body cell dissociation with distinct roles. Sci Rep. 2014 Dec 18;4:7535. PMID: 25520167; PMCID: PMC4269897.
CoinFLP technology for mosaic screens with RNAi or over-expression
Bosch JA, Tran NH, Hariharan IK. CoinFLP: a system for efficient mosaic screening and for visualizing clonal boundaries in Drosophila. Development. 2015 Feb 1;142(3):597-606. PMID: 25605786; PMCID: PMC4302991.
From the abstract: "... The CoinFLP system described here enables mosaic screens in the context of gene knockdown or overexpression by automatically generating a reliable ratio of mutant to wild-type tissue in a developmentally controlled manner. CoinFLP-Gal4 generates mosaic tissues composed of clones of which only a subset expresses Gal4. ..."
From the abstract: "... The CoinFLP system described here enables mosaic screens in the context of gene knockdown or overexpression by automatically generating a reliable ratio of mutant to wild-type tissue in a developmentally controlled manner. CoinFLP-Gal4 generates mosaic tissues composed of clones of which only a subset expresses Gal4. ..."
Arthropod geonimcs symposium--registration is open
I recently received an announcement that registration is now open for the upcoming 9th Annual Arthropod Genomics Symposium and IGT-RCN workshop. The event will be June 17 to 19, 2015, at the K-State Alumni Center, KSU, Manhattan, KS. Symposium Website: http://www.ksu.edu/agc
Wednesday, February 4, 2015
in vivo RNAi screen -- border cell migration
Luo J, Zuo J, Wu J, Wan P, Kang D, Xiang C, Zhu H, Chen J. In vivo RNAi screen identifies candidate signaling genes required for collective cell migration in Drosophila ovary. Sci China Life Sci. 2014 Dec 20. PMID: 25528253.
Monday, February 2, 2015
Review of "breakthroughs" in understanding host-pathogen interactions revealed using RNAi screens
Abnave P, Conti F, Torre C, Ghigo E. What RNAi screens in model organisms revealed about microbicidal response in mammals? Front Cell Infect Microbiol. 2015 Jan 12;4:184. eCollection 2014. PMID: 25629007; PMCID: PMC4290690.
From the abstract: "... The emergence of RNA interference (RNAi) and the ability to apply it toward studies in model organisms have allowed a breakthrough in the elucidation of host-pathogen interactions. The aim of this mini-review is to highlight and describe recent breakthroughs in the field of host-pathogen interactions using RNAi screens of model organisms. We will focus specifically on the model organisms Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio."
From the abstract: "... The emergence of RNA interference (RNAi) and the ability to apply it toward studies in model organisms have allowed a breakthrough in the elucidation of host-pathogen interactions. The aim of this mini-review is to highlight and describe recent breakthroughs in the field of host-pathogen interactions using RNAi screens of model organisms. We will focus specifically on the model organisms Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio."
Thursday, January 29, 2015
CRISPR-based gene photoactivation system
Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M. CRISPR-Cas9-based Photoactivatable Transcription System. Chem Biol. 2015 Jan 20. pii: S1074-5521(14)00459-1. PMID: 25619936.
From the abstract: "... Here we created a light-inducible, user-defined, endogenous gene activation system based on CRISPR-Cas9. ... this ... system can allow rapid and reversible targeted gene activation by light."
From the abstract: "... Here we created a light-inducible, user-defined, endogenous gene activation system based on CRISPR-Cas9. ... this ... system can allow rapid and reversible targeted gene activation by light."
Friday, January 9, 2015
Over-expression of transcription factors--resource and screen paper from Deplancke lab
Early online at Genome Research: A large-scale, in vivo transcription factor screen defines bivalent chromatin as a key property of regulatory factors mediating Drosophila wing development. Claus Schertel, Monica Albarca, Claudia Rockel-Bauer, Nicholas W Kelley, Johannes Bischof, Korneel Hens, Erik van Nimwegen, Konrad Basler and Bart Deplancke. 2015.
From the abstract: "... We generated 596 site-directed transgenic Drosophila lines that contain integrations of individual UAS-TF constructs to facilitate spatio-temporally controlled misexpression in vivo. All transgenes were expressed in the developing wing and two thirds induced specific phenotypic defects. In vivo knock-down of the same genes yielded a phenotype for 50%, with both methods indicating a great potential for misexpression to characterize novel functions in wing growth, patterning and development. Thus, our UAS-TF library provides an important addition to the genetic toolbox of Drosophila research ..."
From the abstract: "... We generated 596 site-directed transgenic Drosophila lines that contain integrations of individual UAS-TF constructs to facilitate spatio-temporally controlled misexpression in vivo. All transgenes were expressed in the developing wing and two thirds induced specific phenotypic defects. In vivo knock-down of the same genes yielded a phenotype for 50%, with both methods indicating a great potential for misexpression to characterize novel functions in wing growth, patterning and development. Thus, our UAS-TF library provides an important addition to the genetic toolbox of Drosophila research ..."
Thursday, January 8, 2015
SignedPPI included in list of "Signaling Breakthroughs of the Year"
How cool is this? SignedPPI, which was developed by the Perrimon lab and DRSC, made it onto the Signaling Breakthroughs of the Year list at Science Signaling!
Here's the paper that describes SignedPPI (free in PubMed Central): Vinayagam A, Zirin J, Roesel C, Hu Y, Yilmazel B, Samsonova AA, Neumüller RA, Mohr SE, Perrimon N. Integrating protein-protein interaction networks with phenotypes reveals signs of interactions. Nat Methods. 2014 Jan;11(1):94-9. PMID: 24240319; PMCID: PMC3877743.
Here's the paper that describes SignedPPI (free in PubMed Central): Vinayagam A, Zirin J, Roesel C, Hu Y, Yilmazel B, Samsonova AA, Neumüller RA, Mohr SE, Perrimon N. Integrating protein-protein interaction networks with phenotypes reveals signs of interactions. Nat Methods. 2014 Jan;11(1):94-9. PMID: 24240319; PMCID: PMC3877743.
NIH funding announcement--basic research in neuroscience
NIH FOA PAS-15-029 is specifically slated for basic research R01 applications and thus, might be of special interest to some of you. NINDS, NIA, NIDA and NIMH are participating.
From the Purpose Statement: "The goal of this Funding Opportunity Announcement (FOA) is to stimulate research addressing fundamental questions in basic neuroscience. Proposed projects can address any area of neuroscience within the missions of the participating institutes and should focus on understanding the structure and/or function of the normal nervous system."
From the Purpose Statement: "The goal of this Funding Opportunity Announcement (FOA) is to stimulate research addressing fundamental questions in basic neuroscience. Proposed projects can address any area of neuroscience within the missions of the participating institutes and should focus on understanding the structure and/or function of the normal nervous system."
Tuesday, January 6, 2015
Sorting by size--with automation
In the "posting this because it looks cool" department:
The FlyCatwalk: A High Throughput Feature-Based Sorting System for Artificial Selection in Drosophila. Vasco Medici, Sibylle Chantal Vonesch, Steven N. Fry, and Ernst Hafen G3 early online January 2, 2015, doi:10.1534/g3.114.013664 http://www.g3journal.org/content/early/2015/01/02/g3.114.013664.abstract
Fig. 1 includes a photo of their device, "a fully automated, high throughput system to sort live fruit flies ... based on morphometric traits."
The FlyCatwalk: A High Throughput Feature-Based Sorting System for Artificial Selection in Drosophila. Vasco Medici, Sibylle Chantal Vonesch, Steven N. Fry, and Ernst Hafen G3 early online January 2, 2015, doi:10.1534/g3.114.013664 http://www.g3journal.org/content/early/2015/01/02/g3.114.013664.abstract
Fig. 1 includes a photo of their device, "a fully automated, high throughput system to sort live fruit flies ... based on morphometric traits."
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