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."

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.

Monday, June 19, 2017

Kinome RNAi screen reported in G3

Parsons LM, Grzeschik NA, Amaratunga K, Burke P, Quinn LM, Richardson HE. A Kinome RNAi Screen in Drosophila Identifies Novel Genes Interacting with Lgl, aPKC and Crb Cell Polarity Genes in Epithelial Tissues. G3 (Bethesda). 2017 Jun 13. PMID: 28611255.

From the abstract: "... To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, we screened a boutique collection of RNAi stocks targeting the kinome for their capacity to modify Drosophila 'cell polarity' eye and wing phenotypes. ... We next conducted a secondary screen to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, JNK, and Notch signalling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways, not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras and lipid/Phospho-inositol-3-kinase (PI3K) signalling pathways. Additionally, we demonstrated that the 'nutrient sensing' kinases, Salt Inducible Kinase 2 and 3 (SIK2 and 3) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. ..."

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."

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. ..."