1 / 19

Genome-Wide RNAi Analysis of Growth and Viability in Drosophila Cells

Genome-Wide RNAi Analysis of Growth and Viability in Drosophila Cells. Boutros et al. Overview. Aim : functional analysis of predicted genes after whole genome sequencing Application : RNAi screen* Quatitative assay of cell number** Results :

nicole
Download Presentation

Genome-Wide RNAi Analysis of Growth and Viability in Drosophila Cells

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Genome-Wide RNAi Analysis of Growth and Viability inDrosophila Cells Boutros et al.

  2. Overview • Aim: • functional analysis of predicted genes after whole genome sequencing • Application: • RNAi screen* • Quatitative assay of cell number** • Results: • characterize the function of 91% of predicted Drosophila genes in cell growth and viability* • Identify genes of known and uncharacterized functions demonstrate the role for the homolog of a mammalian acute myeloid leukemia gene (AML1) in cell survival **

  3. Drosophila • Model organism • Studies • Development • Cell Biology • Population genetics • Signal transduction • Gene regulation and function • Conserved pathways with important roles from flies to humans

  4. RNA interference (RNAi) • Use for: • Idetification of gene function and respective protein • Drosophila cells • dsRNA treatment lead to: • Depletion of corresponding transcript • Loss-of-function phenotypic analysis • Faster and effective way to turn off genes. • Development of new drugs capable of turning off disease causing genes

  5. Experimental approach for genome-wide RNAi screens. • 21,306 primers pairs used for: • Amplification of gene-specific fragments used for synthesis of dsRNAs • dsRNA library targets all genes in the Drosophila genome

  6. Quantitative Assay • Reduction of signal to dying cells • RNAi of the D. melanogaster apoptosis 1 inhibitorD-IAP1 • Left: Luciferous activity indicate ATP levels correlated with the number of Drosophila cells • Right: Treatment of green fluorescencent protein (gfp) dsRNA targeting D-IAP1 induced time-dependent decrease in cell viability

  7. Flourescence Microscopy • Cells after 3 days RNAi • Treatment of D-IAP1 RNAi compared to control dsRNAs • More dying cells with D-IAP1 by the ratio of SYTOX green-labeled nuclei vs. Hoeschst 33342-labeled nuclei.

  8. Genome-wide RNAi screen • 5 days dsRNA treatment • Two embryotic hemocyte (blood cell) lines (Kc187 and S2R+) • 77,880 RNAi experiments

  9. Reproducibility • Screens reproducible • Two independent RNAi screen • Phenotypes with similar z scores • Correlation coefficient=0.86 • Z score- severity or rank of specific RNAi phenotypes

  10. Quantitative RNAi phenotypes of genes • Gray bars- averaged RNAi phenotypes of 72 genes encoding all annotated ribosomal proteins tested • White bars- gfp dsRNA are the negative controls • Black bars- D-IAP1 dsRNAare the more severe phenotypes. Used as the positive control

  11. Frequency of RNAi Phenotypes • Reduced cell number • Z greater of equal to 3 • Threshold shows severity of z score • Defects • cell growth • cell survival • 20% of identified genes has associated mutant alleles with Drosophila • Roles in: • Cell growth • Cell cycle • Anti-apoptotic cell survival

  12. Frequency of Functional Groups • Distribution of most abundant domain predicted gene functions differed with the quatitative severity of the RNAi phenotypes

  13. Classification of Quantitative RNAi phenotypes • Duplicate screens per cell types • Identification of: • Serpent, srp • CG11700-ubiquitin-like gene • Protein degradation • CG15455- AML1-like Transcription factor • Acute myeloid leukemia gene • Oncogene • Encodes transcriptional factors

  14. Classification of Quantitative RNAi Phenotypes Cont’d

  15. Flow Cytometry Analysis • Scans single cells flowing past excitation sources in a liquid medium • Measure fluorescence intensity produced by fluorescent-labeled antibiodies and ligands that bind specific cell-associated molecules • Propidium Iodide stained DNA after 3 days RNAi • Decrease cell size and DNA content

  16. Proportion of Apoptotic Cells (TUNEL) • Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling (TUNEL) • Terminal Transerase labeled DNA breaks • Severe RNAi phenotypes distinguished with dsRNA treatment • 95% treated with dsRNa to CG11700 or DIAP1 • 20% treated with dsRNA to CG15455

  17. Epistasis Analysis • Pan-caspase inbitor (z-VAD-fmk) reverted cell death in response to • RNAi of D-IAP1 and CG11700 • CG15455 and other transcription factor at a lesser extent

  18. Results • Proteome comparison • Percentage of ortholog found for the genes with RNAi viability phenotypes was High • 50 genes had homology to human disease genes • 10 genes implicated in blood-cell leukemia (AML-1) • Genes with antiapopototic functions (FOXOA1 AND MLK) • CG11700 may act in the same pathway as D-IAP1 • Directly preventing Nc caspase-activated apoptotic cell death. • CG15455 and set of TFs may regulate complex responses for cell fate, proliferation, and/or cell survivial

More Related