Zhou J, Xu L, Duan X, Liu W, Zhao X, Wang X, Shang W, Fang X, Yang H, Jia L, Bai J, Zhao J, Wang L, Tong C. Large-scale RNAi screen identified Dhpr as a regulator of mitochondrial morphology and tissue homeostasis. Sci Adv. 2019 Sep 18;5(9):eaax0365. PubMed PMID: 31555733; PubMed Central PMCID: PMC6750926.
Umer Z, Akhtar J, Khan MHF, Shaheen N, Haseeb MA, Mazhar K, Mithani A, Anwar S, Tariq M. Genome-wide RNAi screen in Drosophila reveals Enok as a novel trithorax group regulator. Epigenetics Chromatin. 2019 Sep 23;12(1):55. PubMed PMID: 31547845.
Showing posts with label in vivo assay development. Show all posts
Showing posts with label in vivo assay development. Show all posts
Monday, September 30, 2019
Monday, August 12, 2019
in vivo RNAi screen in the wing
Rotelli MD, Bolling AM, Killion AW, Weinberg AJ, Dixon MJ, Calvi BR. An RNAi Screen for Genes Required for Growth of Drosophila Wing Tissue. G3 (Bethesda). 2019 Aug 6. pii: g3.400581.2019. doi: 10.1534/g3.119.400581. PubMed PMID: 31387856.
Abstract: "Cell division and tissue growth must be coordinated with development. Defects in these processes are the basis for a number of diseases, including developmental malformations and cancer. We have conducted an unbiased RNAi screen for genes that are required for growth in the Drosophila wing, using GAL4-inducible short hairpin RNA (shRNA) fly strains made by the Drosophila RNAi Screening Center. shRNA expression down the center of the larval wing disc using dpp-GAL4, and the central region of the adult wing was then scored for tissue growth and wing hair morphology. Out of 4,753 shRNA crosses that survived to adulthood, 18 had impaired wing growth. FlyBase and the new Alliance of Genome Resources knowledgebases were used to determine the known or predicted functions of these genes and the association of their human orthologs with disease. The function of eight of the genes identified has not been previously defined in Drosophila. The genes identified included those with known or predicted functions in cell cycle, chromosome segregation, morphogenesis, metabolism, steroid processing, transcription, and translation. All but one of the genes are similar to those in humans, and many are associated with disease. Knockdown of lin-52, a subunit of the Myb-MuvB transcription factor, or βNACtes6, a gene involved in protein folding and trafficking, resulted in a switch from cell proliferation to an endoreplication growth program through which wing tissue grew by an increase in cell size (hypertrophy). It is anticipated that further analysis of the genes that we have identified will reveal new mechanisms that regulate tissue growth during development."
Abstract: "Cell division and tissue growth must be coordinated with development. Defects in these processes are the basis for a number of diseases, including developmental malformations and cancer. We have conducted an unbiased RNAi screen for genes that are required for growth in the Drosophila wing, using GAL4-inducible short hairpin RNA (shRNA) fly strains made by the Drosophila RNAi Screening Center. shRNA expression down the center of the larval wing disc using dpp-GAL4, and the central region of the adult wing was then scored for tissue growth and wing hair morphology. Out of 4,753 shRNA crosses that survived to adulthood, 18 had impaired wing growth. FlyBase and the new Alliance of Genome Resources knowledgebases were used to determine the known or predicted functions of these genes and the association of their human orthologs with disease. The function of eight of the genes identified has not been previously defined in Drosophila. The genes identified included those with known or predicted functions in cell cycle, chromosome segregation, morphogenesis, metabolism, steroid processing, transcription, and translation. All but one of the genes are similar to those in humans, and many are associated with disease. Knockdown of lin-52, a subunit of the Myb-MuvB transcription factor, or βNACtes6, a gene involved in protein folding and trafficking, resulted in a switch from cell proliferation to an endoreplication growth program through which wing tissue grew by an increase in cell size (hypertrophy). It is anticipated that further analysis of the genes that we have identified will reveal new mechanisms that regulate tissue growth during development."
Tuesday, April 30, 2019
in vivo RNAi screen for metabolic enzymes required for eye development
Rose C. Pletcher, Sara L. Hardman, Sydney F. Intagliata, Rachel L. Lawson, Aumunique Page and Jason M. Tennessen
A Genetic Screen Using the Drosophila melanogaster TRiP RNAi Collection To Identify Metabolic Enzymes Required for Eye Development
G3: GENES, GENOMES, GENETICS Early online April 29, 2019; https://doi.org/10.1534/g3.119.400193
http://www.g3journal.org/content/early/2019/04/29/g3.119.400193
Abstract: "The metabolic enzymes that compose glycolysis, the citric acid cycle, and other pathways within central carbon metabolism have emerged as key regulators of animal development. These enzymes not only generate the energy and biosynthetic precursors required to support cell proliferation and differentiation, but also moonlight as regulators of transcription, translation, and signal transduction. Many of the genes associated with animal metabolism, however, have never been analyzed in a developmental context, thus highlighting how little is known about the intersection of metabolism and development. Here we address this deficiency by using the Drosophila TRiP RNAi collection to disrupt the expression of over 1,100 metabolism-associated genes within cells of the eye imaginal disc. Our screen not only confirmed previous observations that oxidative phosphorylation serves a critical role in the developing eye, but also implicated a host of other metabolic enzymes in the growth and differentiation of this organ. Notably, our analysis revealed a requirement for glutamine and glutamate metabolic processes in eye development, thereby revealing a role of these amino acids in promoting Drosophila tissue growth. Overall, our analysis highlights how the Drosophila eye can serve as a powerful tool for dissecting the relationship between development and metabolism."
A Genetic Screen Using the Drosophila melanogaster TRiP RNAi Collection To Identify Metabolic Enzymes Required for Eye Development
G3: GENES, GENOMES, GENETICS Early online April 29, 2019; https://doi.org/10.1534/g3.119.400193
http://www.g3journal.org/content/early/2019/04/29/g3.119.400193
Abstract: "The metabolic enzymes that compose glycolysis, the citric acid cycle, and other pathways within central carbon metabolism have emerged as key regulators of animal development. These enzymes not only generate the energy and biosynthetic precursors required to support cell proliferation and differentiation, but also moonlight as regulators of transcription, translation, and signal transduction. Many of the genes associated with animal metabolism, however, have never been analyzed in a developmental context, thus highlighting how little is known about the intersection of metabolism and development. Here we address this deficiency by using the Drosophila TRiP RNAi collection to disrupt the expression of over 1,100 metabolism-associated genes within cells of the eye imaginal disc. Our screen not only confirmed previous observations that oxidative phosphorylation serves a critical role in the developing eye, but also implicated a host of other metabolic enzymes in the growth and differentiation of this organ. Notably, our analysis revealed a requirement for glutamine and glutamate metabolic processes in eye development, thereby revealing a role of these amino acids in promoting Drosophila tissue growth. Overall, our analysis highlights how the Drosophila eye can serve as a powerful tool for dissecting the relationship between development and metabolism."
RNAi screen for genes in escort cells related to germ cell maintenance and differentiation
Gao Y, Mao Y, Xu RG, Zhu R, Zhang M, Sun J, Shen D, Peng P, Xie T, Ni JQ. Defining gene networks controlling the maintenance and function of the differentiation niche by an in vivo systematic RNAi screen. J Genet Genomics. 2019 Jan 20;46(1):19-30. PMID: 30745214.
Abstract: "In the Drosophila ovary, escort cells (ECs) extrinsically control germline stem cell (GSC) maintenance and progeny differentiation. However, the underlying mechanisms remain poorly understood. In this study, we identified 173 EC genes for their roles in controlling GSC maintenance and progeny differentiation by using an in vivo systematic RNAi approach. Of the identified genes, 10 and 163 are required in ECs to promote GSC maintenance and progeny differentiation, respectively. The genes required for progeny differentiation fall into different functional categories, including transcription, mRNA splicing, protein degradation, signal transduction and cytoskeleton regulation. In addition, the GSC progeny differentiation defects caused by defective ECs are often associated with BMP signaling elevation, indicating that preventing BMP signaling is a general functional feature of the differentiation niche. Lastly, exon junction complex (EJC) components, which are essential for mRNA splicing, are required in ECs to promote GSC progeny differentiation by maintaining ECs and preventing BMP signaling. Therefore, this study has identified the major regulators of the differentiation niche, which provides important insights into how stem cell progeny differentiation is extrinsically controlled."
Abstract: "In the Drosophila ovary, escort cells (ECs) extrinsically control germline stem cell (GSC) maintenance and progeny differentiation. However, the underlying mechanisms remain poorly understood. In this study, we identified 173 EC genes for their roles in controlling GSC maintenance and progeny differentiation by using an in vivo systematic RNAi approach. Of the identified genes, 10 and 163 are required in ECs to promote GSC maintenance and progeny differentiation, respectively. The genes required for progeny differentiation fall into different functional categories, including transcription, mRNA splicing, protein degradation, signal transduction and cytoskeleton regulation. In addition, the GSC progeny differentiation defects caused by defective ECs are often associated with BMP signaling elevation, indicating that preventing BMP signaling is a general functional feature of the differentiation niche. Lastly, exon junction complex (EJC) components, which are essential for mRNA splicing, are required in ECs to promote GSC progeny differentiation by maintaining ECs and preventing BMP signaling. Therefore, this study has identified the major regulators of the differentiation niche, which provides important insights into how stem cell progeny differentiation is extrinsically controlled."
Monday, February 11, 2019
Screen explores possible roles of ion channels in Drosophila wing development
George LF, Pradhan SJ, Mitchell D, Josey M, Casey J, Belus MT, Dahal GR, Bates EA. Ion Channel Contributions to Wing Development in Drosophila melanogaster. G3 (Bethesda). 2019 Feb 7. pii: g3.400028.2019. doi: 10.1534/g3.119.400028. PMID: 30733380.
Abstract: "During morphogenesis, cells communicate with each other to shape tissues and organs. Several lines of recent evidence indicate that ion channels play a key role in cellular signaling and tissue morphogenesis. However, little is known about the scope of specific ion-channel types that impinge upon developmental pathways. The Drosophila melanogaster wing is an excellent model in which to address this problem as wing vein patterning is acutely sensitive to changes in developmental pathways. We conducted a screen of 180 ion channels expressed in the wing using loss-of-function mutant and RNAi lines. Here we identify 44 candidates that significantly impacted development of the Drosophila melanogaster wing. Calcium, sodium, potassium, chloride, and ligand-gated cation channels were all identified in our screen, suggesting that a wide variety of ion channel types are important for development. Ion channels belonging to the pickpocket family, the ionotropic receptor family, and the bestrophin family were highly represented among the candidates of our screen. Seven new ion channels with human orthologs that have been implicated in human channelopathies were also identified. Many of the human orthologs of the channels identified in our screen are targets of common general anesthetics, anti-seizure and anti-hypertension drugs, as well as alcohol and nicotine. Our results confirm the importance of ion channels in morphogenesis and identify a number of ion channels that will provide the basis for future studies to understand the role of ion channels in development."
Abstract: "During morphogenesis, cells communicate with each other to shape tissues and organs. Several lines of recent evidence indicate that ion channels play a key role in cellular signaling and tissue morphogenesis. However, little is known about the scope of specific ion-channel types that impinge upon developmental pathways. The Drosophila melanogaster wing is an excellent model in which to address this problem as wing vein patterning is acutely sensitive to changes in developmental pathways. We conducted a screen of 180 ion channels expressed in the wing using loss-of-function mutant and RNAi lines. Here we identify 44 candidates that significantly impacted development of the Drosophila melanogaster wing. Calcium, sodium, potassium, chloride, and ligand-gated cation channels were all identified in our screen, suggesting that a wide variety of ion channel types are important for development. Ion channels belonging to the pickpocket family, the ionotropic receptor family, and the bestrophin family were highly represented among the candidates of our screen. Seven new ion channels with human orthologs that have been implicated in human channelopathies were also identified. Many of the human orthologs of the channels identified in our screen are targets of common general anesthetics, anti-seizure and anti-hypertension drugs, as well as alcohol and nicotine. Our results confirm the importance of ion channels in morphogenesis and identify a number of ion channels that will provide the basis for future studies to understand the role of ion channels in development."
Thursday, November 15, 2018
Thinking about size: in vivo RNAi screen for neuronal genes related to energy homeostasis
Trinh I, Gluscencova OB, Boulianne GL. An in vivo screen for neuronal genes involved in obesity identifies Diacylglycerol kinase as a regulator of insulin secretion. Mol Metab. 2018 Oct 19. pii: S2212-8778(18)30828-7. doi:10.1016/j.molmet.2018.10.006. PMID: 30389349.
From the abstract: "... Here, we have used the genetically tractable fruit fly, Drosophila melanogaster, to identify genes/pathways that function in the nervous system to regulate energy balance. ... We performed an in vivo RNAi screen in Drosophila neurons and assayed for obese or lean phenotypes by measuring changes in levels of stored fats (in the form of triacylglycerides or TAG). Three rounds of screening were performed to verify the reproducibility and specificity of the adiposity phenotypes. Genes that produced >25% increase in TAG (206 in total) underwent a second round of screening to verify their effect on TAG levels by retesting the same RNAi line to validate the phenotype. All remaining hits were screened a third time by testing the TAG levels of additional RNAi lines against the genes of interest to rule out any off-target effects. ... We identified 24 genes including 20 genes that have not been previously associated with energy homeostasis. One identified hit, Diacylglycerol kinase (Dgk), has mammalian homologues that have been implicated in genome-wide association studies for metabolic defects. ... Altogether, we have identified several genes that act within the CNS to regulate energy homeostasis. One of these, Dgk, acts within the insulin-producing cells to regulate the secretion of dILPs and energy homeostasis in Drosophila."
From the abstract: "... Here, we have used the genetically tractable fruit fly, Drosophila melanogaster, to identify genes/pathways that function in the nervous system to regulate energy balance. ... We performed an in vivo RNAi screen in Drosophila neurons and assayed for obese or lean phenotypes by measuring changes in levels of stored fats (in the form of triacylglycerides or TAG). Three rounds of screening were performed to verify the reproducibility and specificity of the adiposity phenotypes. Genes that produced >25% increase in TAG (206 in total) underwent a second round of screening to verify their effect on TAG levels by retesting the same RNAi line to validate the phenotype. All remaining hits were screened a third time by testing the TAG levels of additional RNAi lines against the genes of interest to rule out any off-target effects. ... We identified 24 genes including 20 genes that have not been previously associated with energy homeostasis. One identified hit, Diacylglycerol kinase (Dgk), has mammalian homologues that have been implicated in genome-wide association studies for metabolic defects. ... Altogether, we have identified several genes that act within the CNS to regulate energy homeostasis. One of these, Dgk, acts within the insulin-producing cells to regulate the secretion of dILPs and energy homeostasis in Drosophila."
Where am I? in vivo RNAi screen looks at larval chordotonal organs
Hassan A, Timerman Y, Hamdan R, Sela N, Avetisyan A, Halachmi N, Salzberg A. An RNAi Screen Identifies New Genes Required for Normal Morphogenesis of Larval Chordotonal Organs. G3 (Bethesda). 2018 May 31;8(6):1871-1884. doi: 10.1534/g3.118.200218. PubMed PMID: 29678948; PubMed Central PMCID: PMC5982817.
From the abstract: "The proprioceptive chordotonal organs (ChO) of a fly larva respond to mechanical stimuli generated by muscle contractions and consequent deformations of the cuticle. The ability of the ChO to sense the relative displacement of its epidermal attachment sites likely depends on the correct mechanical properties of the accessory (cap and ligament) and attachment cells that connect the sensory unit (neuron and scolopale cell) to the cuticle. ... Here we describe an RNAi screen that focused on the ChO's accessory and attachment cells and was performed in 2nd instar larvae to allow for phenotypic analysis of ChOs that had already experienced mechanical stresses during larval growth. Nearly one thousand strains carrying RNAi constructs targeting more than 500 candidate genes were screened for their effects on ChO morphogenesis. The screen identified 31 candidate genes whose knockdown within the ChO lineage disrupted various aspects of cell fate determination, cell differentiation, cellular morphogenesis and cell-cell attachment. Most interestingly, one phenotypic group consisted of genes that affected the response of specific ChO cell types to developmental organ stretching, leading to abnormal pattern of cell elongation. The 'cell elongation' group included the transcription factors Delilah and Stripe ... Other genes found to affect the pattern of ChO cell elongation ... represent putative effectors that link between cell-fate determinants and the realization of cell-specific mechanical properties."
From the abstract: "The proprioceptive chordotonal organs (ChO) of a fly larva respond to mechanical stimuli generated by muscle contractions and consequent deformations of the cuticle. The ability of the ChO to sense the relative displacement of its epidermal attachment sites likely depends on the correct mechanical properties of the accessory (cap and ligament) and attachment cells that connect the sensory unit (neuron and scolopale cell) to the cuticle. ... Here we describe an RNAi screen that focused on the ChO's accessory and attachment cells and was performed in 2nd instar larvae to allow for phenotypic analysis of ChOs that had already experienced mechanical stresses during larval growth. Nearly one thousand strains carrying RNAi constructs targeting more than 500 candidate genes were screened for their effects on ChO morphogenesis. The screen identified 31 candidate genes whose knockdown within the ChO lineage disrupted various aspects of cell fate determination, cell differentiation, cellular morphogenesis and cell-cell attachment. Most interestingly, one phenotypic group consisted of genes that affected the response of specific ChO cell types to developmental organ stretching, leading to abnormal pattern of cell elongation. The 'cell elongation' group included the transcription factors Delilah and Stripe ... Other genes found to affect the pattern of ChO cell elongation ... represent putative effectors that link between cell-fate determinants and the realization of cell-specific mechanical properties."
Monday, June 4, 2018
in vivo RNAi screen focused on the female germline
Cho Y, Lai CM, Lin KY, Hsu HJ. A Targeted RNAi Screen Reveals Drosophila Female-Sterile Genes That Control the Size of Germline Stem Cell Niche During Development. G3 (Bethesda). 2018 May 15. PMID: 29764959.
From the abstract: "... we conducted a small-scale RNAi screen of 560 individually expressed UAS-RNAi lines with targets implicated in female fertility. RNAi was expressed in the soma of larval gonads, and screening for reduced egg production and abnormal ovarian morphology was performed in adults. Twenty candidates that affect ovarian development were identified and subsequently knocked down in the soma only during niche formation. ..."
From the abstract: "... we conducted a small-scale RNAi screen of 560 individually expressed UAS-RNAi lines with targets implicated in female fertility. RNAi was expressed in the soma of larval gonads, and screening for reduced egg production and abnormal ovarian morphology was performed in adults. Twenty candidates that affect ovarian development were identified and subsequently knocked down in the soma only during niche formation. ..."
Thursday, May 31, 2018
New in vivo RNAi screen report -- larval chordotonal organ morphogenesis
An RNAi Screen Identifies New Genes Required for Normal Morphogenesis of Larval Chordotonal Organs
Abeer Hassan, Yael Timerman, Rana Hamdan, Nitzan Sela, Adel Avetisyan, Naomi Halachmi and Adi Salzberg
G3: GENES, GENOMES, GENETICS June 1, 2018 vol. 8 no. 6 1871-1884
https://doi.org/10.1534/g3.118.200218
From the abstract: "... Here we describe an RNAi screen that focused on the ChO’s accessory and attachment cells and was performed in 2nd instar larvae to allow for phenotypic analysis of ChOs that had already experienced mechanical stresses during larval growth. Nearly one thousand strains carrying RNAi constructs targeting more than 500 candidate genes were screened for their effects on ChO morphogenesis. The screen identified 31 candidate genes whose knockdown within the ChO lineage disrupted various aspects of cell fate determination, cell differentiation, cellular morphogenesis and cell-cell attachment. ..."
Abeer Hassan, Yael Timerman, Rana Hamdan, Nitzan Sela, Adel Avetisyan, Naomi Halachmi and Adi Salzberg
G3: GENES, GENOMES, GENETICS June 1, 2018 vol. 8 no. 6 1871-1884
https://doi.org/10.1534/g3.118.200218
From the abstract: "... Here we describe an RNAi screen that focused on the ChO’s accessory and attachment cells and was performed in 2nd instar larvae to allow for phenotypic analysis of ChOs that had already experienced mechanical stresses during larval growth. Nearly one thousand strains carrying RNAi constructs targeting more than 500 candidate genes were screened for their effects on ChO morphogenesis. The screen identified 31 candidate genes whose knockdown within the ChO lineage disrupted various aspects of cell fate determination, cell differentiation, cellular morphogenesis and cell-cell attachment. ..."
in vivo fly RNAi screen identifies ortholog of SPO11 DNA break protein as pro-tumor in a fly tumor model
Rossi F, Molnar C, Hashiyama K, Heinen JP, Pampalona J, Llamazares S, Reina J, Hashiyama T, Rai M, Pollarolo G, Fernández-Hernández I, Gonzalez C. An in vivo genetic screen in Drosophila identifies the orthologue of human cancer/testis gene SPO11 among a network of targets to inhibit lethal(3)malignant brain tumour growth. Open Biol. 2017 Aug;7(8). pii: 170156. PMID: 28855394; PMCID: PMC5577452.
The abstract: "Using transgenic RNAi technology, we have screened over 4000 genes to identify targets to inhibit malignant growth caused by the loss of function of lethal(3)malignant brain tumour in Drosophila in vivo. We have identified 131 targets, which belong to a wide range of gene ontologies. Most of these target genes are not significantly overexpressed in mbt tumours hence showing that, rather counterintuitively, tumour-linked overexpression is not a good predictor of functional requirement. Moreover, we have found that most of the genes upregulated in mbt tumours remain overexpressed in tumour-suppressed double-mutant conditions, hence revealing that most of the tumour transcriptome signature is not necessarily correlated with malignant growth. One of the identified target genes is meiotic W68 (mei-W68), the Drosophila orthologue of the human cancer/testis gene Sporulation-specific protein 11 (SPO11), the enzyme that catalyses the formation of meiotic double-strand breaks. We show that Drosophila mei-W68/SPO11 drives oncogenesis by causing DNA damage in a somatic tissue, hence providing the first instance in which a SPO11 orthologue is unequivocally shown to have a pro-tumoural role. Altogether, the results from this screen point to the possibility of investigating the function of human cancer relevant genes in a tractable experimental model organism like Drosophila."
The abstract: "Using transgenic RNAi technology, we have screened over 4000 genes to identify targets to inhibit malignant growth caused by the loss of function of lethal(3)malignant brain tumour in Drosophila in vivo. We have identified 131 targets, which belong to a wide range of gene ontologies. Most of these target genes are not significantly overexpressed in mbt tumours hence showing that, rather counterintuitively, tumour-linked overexpression is not a good predictor of functional requirement. Moreover, we have found that most of the genes upregulated in mbt tumours remain overexpressed in tumour-suppressed double-mutant conditions, hence revealing that most of the tumour transcriptome signature is not necessarily correlated with malignant growth. One of the identified target genes is meiotic W68 (mei-W68), the Drosophila orthologue of the human cancer/testis gene Sporulation-specific protein 11 (SPO11), the enzyme that catalyses the formation of meiotic double-strand breaks. We show that Drosophila mei-W68/SPO11 drives oncogenesis by causing DNA damage in a somatic tissue, hence providing the first instance in which a SPO11 orthologue is unequivocally shown to have a pro-tumoural role. Altogether, the results from this screen point to the possibility of investigating the function of human cancer relevant genes in a tractable experimental model organism like Drosophila."
Wednesday, July 19, 2017
Short vs. long hairpins in fly RNAi
Bartoletti R, Capozzoli B, Moore J, Moran J, Shrawder B, Vivekanand P. Short hairpin RNA is more effective than long hairpin RNA in eliciting pointed loss-of-function phenotypes in Drosophila. Genesis. 2017 Jul;55(7). PMID: 28464429.
From the abstract: "Pointed (Pnt) is a transcriptional activator ... As mutations in pnt perturb the development of several tissues, we wanted to examine the effect and efficacy of using RNAi to target Pnt. We have expressed pnt RNAi in the eyes, oocyte, and heart cells using three different RNAi lines: Valium20, Valium10, and VDRC. Valium20 is distinct since it generates a short hairpin RNA (shRNA), while Valium10 and VDRC produce long hairpin dsRNA. We found that for each tissue examined Valium20 exhibited the strongest phenotype while the Valium10 and VDRC lines produced varying levels of severity and that the long hairpin RNA produced by the Valium10 and VDRC lines are unable to effectively knockdown pnt in embryonic tissues."
From the abstract: "Pointed (Pnt) is a transcriptional activator ... As mutations in pnt perturb the development of several tissues, we wanted to examine the effect and efficacy of using RNAi to target Pnt. We have expressed pnt RNAi in the eyes, oocyte, and heart cells using three different RNAi lines: Valium20, Valium10, and VDRC. Valium20 is distinct since it generates a short hairpin RNA (shRNA), while Valium10 and VDRC produce long hairpin dsRNA. We found that for each tissue examined Valium20 exhibited the strongest phenotype while the Valium10 and VDRC lines produced varying levels of severity and that the long hairpin RNA produced by the Valium10 and VDRC lines are unable to effectively knockdown pnt in embryonic tissues."
Monday, July 10, 2017
Methods papers on in vivo RNAi -- stem cell and germline studies
New methods papers on in vivo fly RNAi:
Riechmann V. In vivo RNAi in the Drosophila Follicular Epithelium: Analysis of Stem Cell Maintenance, Proliferation, and Differentiation. Methods Mol Biol. 2017;1622:185-206. doi: 10.1007/978-1-4939-7108-4_14. PubMed PMID: 28674810.
Blake AJ, Finger DS, Hardy VL, Ables ET. RNAi-Based Techniques for the Analysis of Gene Function in Drosophila Germline Stem Cells. Methods Mol Biol. 2017;1622:161-184. doi: 10.1007/978-1-4939-7108-4_13. PubMed PMID: 28674809.
Riechmann V. In vivo RNAi in the Drosophila Follicular Epithelium: Analysis of Stem Cell Maintenance, Proliferation, and Differentiation. Methods Mol Biol. 2017;1622:185-206. doi: 10.1007/978-1-4939-7108-4_14. PubMed PMID: 28674810.
Blake AJ, Finger DS, Hardy VL, Ables ET. RNAi-Based Techniques for the Analysis of Gene Function in Drosophila Germline Stem Cells. Methods Mol Biol. 2017;1622:161-184. doi: 10.1007/978-1-4939-7108-4_13. PubMed PMID: 28674809.
Monday, June 12, 2017
in vivo RNAi screen explores MANF ortholog DmManf
Lindström R, Lindholm P, Palgi M, Saarma M, Heino TI. In vivo screening reveals interactions between Drosophila Manf and genes involved in the mitochondria and the ubiquinone synthesis pathway. BMC Genet. 2017 Jun 2;18(1):52. PMID: 28578657; PMCID: PMC5455201.
From the abstract: "Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) and Cerebral Dopamine Neurotrophic Factor (CDNF) form an evolutionarily conserved family of neurotrophic factors. Orthologues for MANF/CDNF are the only neurotrophic factors as yet identified in invertebrates with conserved amino acid sequence. ... Here, we have employed the Drosophila genetics to screen for potential interaction partners of Drosophila Manf (DmManf) in vivo. ... In addition to the previously known functions of MANF/CDNF protein family, DmManf also interacts with mitochondria-related genes. Our data supports the functional importance of these evolutionarily significant proteins and provides new insights for the future studies."
From the abstract: "Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) and Cerebral Dopamine Neurotrophic Factor (CDNF) form an evolutionarily conserved family of neurotrophic factors. Orthologues for MANF/CDNF are the only neurotrophic factors as yet identified in invertebrates with conserved amino acid sequence. ... Here, we have employed the Drosophila genetics to screen for potential interaction partners of Drosophila Manf (DmManf) in vivo. ... In addition to the previously known functions of MANF/CDNF protein family, DmManf also interacts with mitochondria-related genes. Our data supports the functional importance of these evolutionarily significant proteins and provides new insights for the future studies."
Wednesday, May 17, 2017
RNAi screen focused on chaperone function in the fly eye
Raut S, Mallik B, Parichha A, V A, Sahi C, Kumar V. RNAi-Mediated Reverse Genetic Screen Identified Drosophila Chaperones Regulating Eye and Neuromuscular Junction Morphology. G3 (Bethesda). 2017 May 12. pii: g3.117.041632. PMID: 28500055.
From the abstract: "Accumulation of toxic proteins in neurons have been linked with the onset of neurodegenerative diseases ... Molecular chaperones help protein folding and resolubilization of unfolded proteins thereby reducing the protein aggregation stress. While most of the chaperones are expressed in neurons, their functional relevance largely remains unknown. Here, using bioinformatics analysis, we identified 95 Drosophila chaperones and classified them into seven different classes. Ubiquitous actin5C-Gal4 mediated RNAi knockdown revealed that about 50% of the chaperones are essential in Drosophila[.] Knocking down these genes in eyes revealed that about 30% of the essential chaperones are crucial for eye development. ..."
From the abstract: "Accumulation of toxic proteins in neurons have been linked with the onset of neurodegenerative diseases ... Molecular chaperones help protein folding and resolubilization of unfolded proteins thereby reducing the protein aggregation stress. While most of the chaperones are expressed in neurons, their functional relevance largely remains unknown. Here, using bioinformatics analysis, we identified 95 Drosophila chaperones and classified them into seven different classes. Ubiquitous actin5C-Gal4 mediated RNAi knockdown revealed that about 50% of the chaperones are essential in Drosophila[.] Knocking down these genes in eyes revealed that about 30% of the essential chaperones are crucial for eye development. ..."
Tuesday, January 24, 2017
RNAi and proteomics study identifies glial genes required for behavior
Ng FS, Sengupta S, Huang Y, Yu AM, You S, Roberts MA, Iyer LK, Yang Y, Jackson FR. TRAP-seq Profiling and RNAi-Based Genetic Screens Identify Conserved Glial Genes Required for Adult Drosophila Behavior. Front Mol Neurosci. 2016 Dec 22;9:146. PMID: 28066175; PMCID: PMC5177635.
From the abstract: "... Glial astrocytes and glia-neuron signaling ... have important modulatory functions in sleep, circadian behavior, memory and plasticity. To better understand mechanisms of glia-neuron signaling in the context of behavior, we have conducted cell-specific, genome-wide expression profiling of adult Drosophila astrocyte-like brain cells and performed RNA interference (RNAi)-based genetic screens ... Glial-specific expression of 653 RNAi constructs targeting 318 genes identified multiple factors associated with altered locomotor activity, circadian rhythmicity and/or responses to mechanical stress (bang sensitivity). ... These results strongly support the idea that glia-neuron communication is vital for adult behavior."
From the abstract: "... Glial astrocytes and glia-neuron signaling ... have important modulatory functions in sleep, circadian behavior, memory and plasticity. To better understand mechanisms of glia-neuron signaling in the context of behavior, we have conducted cell-specific, genome-wide expression profiling of adult Drosophila astrocyte-like brain cells and performed RNA interference (RNAi)-based genetic screens ... Glial-specific expression of 653 RNAi constructs targeting 318 genes identified multiple factors associated with altered locomotor activity, circadian rhythmicity and/or responses to mechanical stress (bang sensitivity). ... These results strongly support the idea that glia-neuron communication is vital for adult behavior."
Friday, December 9, 2016
From the Department of Very Specific Phenotypes: in vivo RNAi screen related to formation of protrusions on the corneal lens of the fly eye
Minami R, Sato C, Yamahama Y, Kubo H, Hariyama T, Kimura KI. An RNAi Screen for Genes Involved in Nanoscale Protrusion Formation on Corneal Lens in Drosophila melanogaster. Zoolog Sci. 2016 Dec;33(6):583-591. PMID: 27927092.
From the abstract: "The "moth-eye" structure, which is observed on the surface of corneal lens in several insects, supports anti-reflective and self-cleaning functions due to nanoscale protrusions ... the mechanism of protrusion formation from cell-secreted substances is unknown. ... In the present study, we sought to identify genes involved in "moth-eye" structure, formation in order to elucidate the developmental mechanism of the protrusions in Drosophila. We re-examined the aberrant patterns in classical glossy-eye mutants by scanning electron microscope and classified the aberrant patterns into groups. Next, we screened genes encoding putative structural cuticular proteins and genes involved in cuticular formation using eye specific RNAi silencing methods combined with the Gal4/UAS expression system. We identified 12 of 100 candidate genes, such as cuticular proteins family genes (Cuticular protein 23B and Cuticular protein 49Ah), cuticle secretion-related genes (Syntaxin 1A and Sec61 ββ subunit), ecdysone signaling and biosynthesis-related genes (Ecdysone receptor, Blimp-1, and shroud), and genes involved in cell polarity/cell architecture (Actin 5C, shotgun, armadillo, discs large1, and coracle). Although some of the genes we identified may affect corneal protrusion formation indirectly through general patterning defects in eye formation, these initial findings have encouraged us to more systematically explore the precise mechanisms underlying the formation of nanoscale protrusions in Drosophila."
From the abstract: "The "moth-eye" structure, which is observed on the surface of corneal lens in several insects, supports anti-reflective and self-cleaning functions due to nanoscale protrusions ... the mechanism of protrusion formation from cell-secreted substances is unknown. ... In the present study, we sought to identify genes involved in "moth-eye" structure, formation in order to elucidate the developmental mechanism of the protrusions in Drosophila. We re-examined the aberrant patterns in classical glossy-eye mutants by scanning electron microscope and classified the aberrant patterns into groups. Next, we screened genes encoding putative structural cuticular proteins and genes involved in cuticular formation using eye specific RNAi silencing methods combined with the Gal4/UAS expression system. We identified 12 of 100 candidate genes, such as cuticular proteins family genes (Cuticular protein 23B and Cuticular protein 49Ah), cuticle secretion-related genes (Syntaxin 1A and Sec61 ββ subunit), ecdysone signaling and biosynthesis-related genes (Ecdysone receptor, Blimp-1, and shroud), and genes involved in cell polarity/cell architecture (Actin 5C, shotgun, armadillo, discs large1, and coracle). Although some of the genes we identified may affect corneal protrusion formation indirectly through general patterning defects in eye formation, these initial findings have encouraged us to more systematically explore the precise mechanisms underlying the formation of nanoscale protrusions in Drosophila."
Tuesday, November 1, 2016
Methods reviews for in vivo RNAi screening in Drosophila
Four! RNAi-relevant methods papers in the recent Drosophila-focused issue of Methods in Molecular Biology:
Billmann M, Boutros M. Methods for High-Throughput RNAi Screening in Drosophila Cells. Methods Mol Biol. 2016;1478:95-116. PMID: 27730577.
Kaya-Çopur A, Schnorrer F. A Guide to Genome-Wide In Vivo RNAi Applications in Drosophila. Methods Mol Biol. 2016;1478:117-143. PMID: 27730578.
Zhou J, Tong C. Design and Methods of Large-Scale RNA Interference Screens in Drosophila. Methods Mol Biol. 2016;1470:163-9. PMID: 27581292. Chen X, Xu L.
Genome-Wide RNAi Screening to Dissect the TGF-β Signal Transduction Pathway. Methods Mol Biol. 2016;1344:365-77. PMID: 26520138.
Billmann M, Boutros M. Methods for High-Throughput RNAi Screening in Drosophila Cells. Methods Mol Biol. 2016;1478:95-116. PMID: 27730577.
Kaya-Çopur A, Schnorrer F. A Guide to Genome-Wide In Vivo RNAi Applications in Drosophila. Methods Mol Biol. 2016;1478:117-143. PMID: 27730578.
Zhou J, Tong C. Design and Methods of Large-Scale RNA Interference Screens in Drosophila. Methods Mol Biol. 2016;1470:163-9. PMID: 27581292. Chen X, Xu L.
Genome-Wide RNAi Screening to Dissect the TGF-β Signal Transduction Pathway. Methods Mol Biol. 2016;1344:365-77. PMID: 26520138.
Monday, October 31, 2016
RNAi used to induce ROS stress in a study of oogenesis
Perkins AT, Das TM, Panzera LC, Bickel SE. Oxidative stress in oocytes during midprophase induces premature loss of cohesion and chromosome segregation errors. Proc Natl Acad Sci U S A. 2016 Oct 17. pii: 201612047. PMID: 27791141.
From the abstract: "... A growing body of evidence suggests that meiotic cohesion deteriorates as oocytes age ... One hallmark of aging cells is an increase in oxidative damage caused by reactive oxygen species (ROS). Therefore, increased oxidative damage in older oocytes may be one of the factors that leads to premature loss of cohesion and segregation errors. To test this hypothesis, we used an RNAi strategy to induce oxidative stress in Drosophila oocytes and measured the fidelity of chromosome segregation during meiosis. ..."
From the abstract: "... A growing body of evidence suggests that meiotic cohesion deteriorates as oocytes age ... One hallmark of aging cells is an increase in oxidative damage caused by reactive oxygen species (ROS). Therefore, increased oxidative damage in older oocytes may be one of the factors that leads to premature loss of cohesion and segregation errors. To test this hypothesis, we used an RNAi strategy to induce oxidative stress in Drosophila oocytes and measured the fidelity of chromosome segregation during meiosis. ..."
Tick tock! Screening the clock.
Agrawal P, Hardin PE. An RNAi Screen to Identify Protein Phosphatases that Function Within the Drosophila Circadian Clock. G3 (Bethesda). 2016 Oct 26. PMID: 27784754.
From the abstract: "Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. ... To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. ... Additional RNAi lines, transposon inserts, overexpression and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. ... 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans."
From the abstract: "Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. ... To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. ... Additional RNAi lines, transposon inserts, overexpression and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. ... 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans."
Tuesday, September 6, 2016
Methods paper -- in vivo RNAi screens
Zhou J, Tong C. Design and Methods of Large-Scale RNA Interference Screens in Drosophila. Methods Mol Biol. 2016;1470:163-9. PMID: 27581292.
From the abstract: "... Here, we discuss methods for the design and performance of a large-scale in vivo RNAi screen in Drosophila. Furthermore, methods for the validation of results and analysis of data will be introduced."
From the abstract: "... Here, we discuss methods for the design and performance of a large-scale in vivo RNAi screen in Drosophila. Furthermore, methods for the validation of results and analysis of data will be introduced."
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