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

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D145: Genomics and Proteomics

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

    1. 1. Genome-Wide RNAi Analysis of Growth and Viability in Drosophila Cells Michael Boutros, Amy A. Kiger, Susan Armknecht, Kim Kerr, Marc Hild, Britta Koch, Stefan A. Haas, Heidelberg Fly Array Consortium, Renato Paro, Norbert Perrimon (2004) Presenters: Lian Lim Luigi Leung
    2. 2. History of RNAi <ul><li>Rich Jorgensen (1986) </li></ul><ul><ul><li>asked to create a unique flower to gain funding for new gentech company </li></ul></ul><ul><ul><li>wanted to create extremely purple Petunia </li></ul></ul><ul><li>Thought: add more copies of the gene  more purple </li></ul><ul><li>Got: White petunia! </li></ul><ul><li>10 years to figure out </li></ul><ul><li>why this happened </li></ul>
    3. 3. RNAi and the Nobel Prize <ul><li>Craig Melo & Andre Fire (1996) </li></ul><ul><li>Injected dsRNA (RNAi) into C. Elegans and silenced target gene </li></ul><ul><li>First to define this cause of gene silencing </li></ul><ul><ul><li>Coined term RNAi (RNA interference) </li></ul></ul><ul><li>Won Nobel Prize in Physiology of Medicine in 2006 </li></ul>
    4. 4. <ul><li>Dicer (enzyme) binds dsRNA </li></ul><ul><li>Dicer cuts dsRNA into short interfering RNAs (siRNA) </li></ul><ul><li>RISC (RNA- Induced Silencing Complex) binds to siRNA and finds complementary mRNA </li></ul><ul><li>RISC cleaves mRNA </li></ul><ul><ul><li>Gene is knocked down </li></ul></ul>Technicalities
    5. 5. Purpose of Study <ul><li>Used Drosophila as model organism </li></ul><ul><li>Genome sequenced so now we need to find functions of genes </li></ul><ul><li>Use RNAi screens to determine the functions of all genes (91%) in Drosophila </li></ul><ul><ul><li>Genes for cell growth and viability </li></ul></ul>
    6. 6. Methods <ul><li>Knock down gene </li></ul><ul><ul><li>Using RNAi </li></ul></ul><ul><li>Observe cells </li></ul><ul><ul><li>With fluorescence probes </li></ul></ul><ul><li>Calculate Z-score and compare </li></ul><ul><ul><li>Deduce function of uncharacterized known genes </li></ul></ul>
    7. 7. Methods/Results - Observe cells <ul><li>Luciferase </li></ul><ul><ul><li>Luciferase activity ∝ ATP level </li></ul></ul><ul><li>gfp (green fluorescence protein) </li></ul><ul><ul><li>tagged cells ∝ alive </li></ul></ul>
    8. 8. Methods/Results - Observe cells <ul><li>Fluorescence microscopy (of cells after 3 days RNAi) </li></ul><ul><ul><li>D-IAP1 (inhibitor of apoptosis) (+ve control) </li></ul></ul><ul><ul><li>ratio (control) </li></ul></ul>
    9. 9. Methods/Results - Observe cells <ul><li>Genome wide RNAi screen </li></ul><ul><ul><li>(cells after 5 days dsRNA) </li></ul></ul><ul><li>4 control wells: </li></ul><ul><li>D-IAP1 </li></ul><ul><li>Gfp </li></ul><ul><li>Rho1 </li></ul><ul><li>no dsRNA </li></ul>
    10. 10. Z-score Transformation <ul><li>“ converting to Z-score” </li></ul><ul><li>Standardization </li></ul><ul><ul><li>Transforming scores on a variable to Z-scores </li></ul></ul>Cheadle, C., Vawter, M., Freed, W., Becker, K. “Analysis of Microarray Data Using Z Score Transformation.” J Mol Diagn 5.2 (2003 May): 73-81
    11. 11. Methods/Results - Observe cells <ul><li>Double check </li></ul><ul><ul><li>reproducible phenotype </li></ul></ul><ul><li>Consistency </li></ul>
    12. 12. Methods/Results - Observe cells <ul><li>z-scores: </li></ul><ul><ul><li>gfp </li></ul></ul><ul><ul><ul><li>(negative control) </li></ul></ul></ul><ul><ul><li>Ribosomal prot. (72 genes) </li></ul></ul><ul><ul><li>D-IAP1 </li></ul></ul><ul><ul><ul><li>(positive control) </li></ul></ul></ul>
    13. 13. Methods/Results - Observe cells <ul><li>Distribution of z-score freq. of RNAi phenotype </li></ul><ul><li>Select genes for analysis </li></ul>
    14. 14. Methods/Results - Observe cells (z-score >3)
    15. 15. Methods/Results - Observe cells <ul><li>Similar z-score </li></ul><ul><li>infer similar function. </li></ul>
    16. 16. Methods/Results - Further Analysis <ul><li>2 genes for further analysis </li></ul><ul><ul><li>CG11700 </li></ul></ul><ul><ul><ul><li>(ubiquitin-like gene) </li></ul></ul></ul><ul><ul><li>CG15455 </li></ul></ul><ul><ul><ul><li>(AML1 transcription factor) </li></ul></ul></ul>
    17. 17. Method - Flow Cytometry
    18. 18. Methods/Results - Further Analysis <ul><li>Flow cytometry </li></ul>
    19. 19. Method - TUNEL <ul><li>TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) (TdT-mediated dUTP nick-end labeling) </li></ul>
    20. 20. Methods/Results - Further Analysis <ul><li>TUNEL ((TdT-mediated dUTP nick-end labeling) </li></ul>
    21. 21. Methods - Rescue <ul><li>Previously Nc caspase = directly inhibited by D-IAPI =>prevent apoptosis </li></ul>
    22. 22. Methods/Results - Further Analysis <ul><li>Rescue of RNAi growth + viability phenotypes </li></ul>
    23. 23. Recap <ul><li>aim = function of nearly all predicted genes in Drosophila </li></ul><ul><li>Knock down gene </li></ul><ul><ul><li>Using RNAi </li></ul></ul><ul><li>Observe cells </li></ul><ul><ul><li>With fluorescence probes </li></ul></ul><ul><li>Z-score transformation -> compare/group Z-score </li></ul><ul><ul><li>Deduce function of uncharacterized known genes </li></ul></ul>
    24. 24. Importance of the Study <ul><li>Study uncovered common key regulators for animal cell survival and proliferation </li></ul><ul><ul><li>CG11700 may act in same pathway as D-IAP1 </li></ul></ul><ul><li>Comparison of severe RNAi phenotypes (z > 5) to yeast and animal homologs suggest that metazoans have specific mechanisms to maintain cell viability </li></ul>
    25. 25. Importance of RNAi <ul><li>Determination of unknown and evolutionarily conserved gene functions </li></ul><ul><li>Sensitive way to detect gene functions </li></ul><ul><li>Understand more complex gene functions via statistical clustering </li></ul><ul><li>Adaptable to screen many different cellular pathways </li></ul>
    26. 26. Further Reading RNAi – A powerful tool to unravel hepatitis C virus–host interactions within the infectious life cycle Joachim Lupberger, Laurent Brino, Thomas F. Baumert Focusing on RISC assembly in mammalian cells Junmei Hong, Na Wei, Alistair Chalk, Jue Wang, Yutong Song, Fan Yi, Ren-Ping Qiao, Erik L.L. Sonnhammer, Claes Wahlestedt, Zicai Liang, Quan Du Analysis of Microarray Data Using Z Score Transformation Cheadle, C., Vawter, M., Freed, W., Becker, K.
    27. 27. More on RNAi <ul><li>Cops and Pirates analogy for RNAi and Viruses </li></ul><ul><li>Cops search and destroy </li></ul><ul><ul><li>Destroy viral RNA and anything similar to </li></ul></ul><ul><ul><li>http://www.youtube.com/watch?v=H5udFjWDM3E </li></ul></ul>
    28. 28. Questions/Comments

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