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."
Tuesday, November 24, 2015
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.
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