Presentation on theme: "1 PI: Dr. James C.-K. Shen ( 沈哲鯤 ) Co-PI: Dr. Michael Hsiao ( 蕭宏昇副研究員 ) Co-PI/ Manager: Dr. King-Song Jeng ( 鄭金松副技師 ) Speaker: King-Song Jeng ( 鄭金松 ) Institute."— Presentation transcript:
1 PI: Dr. James C.-K. Shen ( 沈哲鯤 ) Co-PI: Dr. Michael Hsiao ( 蕭宏昇副研究員 ) Co-PI/ Manager: Dr. King-Song Jeng ( 鄭金松副技師 ) Speaker: King-Song Jeng ( 鄭金松 ) Institute of Molecular Biology / Genomic Research Center Academia Sinica Sponsored by National Research Program for Genomic Medicine, National Science Council / Academia Sinica C6-The RNAi Core 10/16/98
2 Outline The RNAi Consortium (TRC) The RNAi Consortium (TRC) Current status of the RNAi Core Current status of the RNAi Core Collaboration Research Project Collaboration Research Project
3 The RNAi Consortium (TRC) Objectives: Create genome wide, renewable RNAi reagents for research and educational uses; Develop, validate and optimize materials, methodologies and information for their effective application in research.
4 The RNAi Consortium (TRC) The RNAi Core Phase I (May/2004 to Apr/2007)Jun/2005 to Apr/2008 Phase II (Oct/2007 to Sept/2011) May/2008 to Apr/2011 Phases of TRC Program
5 Sponsoring MembersCollaborating Laboratories Taiwan, ROC + Companies in USA:TRC-I: – Bristol-Myers Squibb – Eli Lilly – Novartis – Sigma – Aldrich TRC-II: – Broad Institute of MIT/ Harvard – Ontario Institute for Cancer Research – Bristol-Myers Squibb – Sigma – Aldrich 1. Nir Hacohen, Whitehead Institute, HMS, Massachusetts General Hospital 2. David Sabatini, Whitehead Institute/MIT 3. Stuart Schreiber, Harvard University, Broad Institute 4. Sheila Stewart, Washington University 5. Brent Stockwell, Whitehead Institute 6. David Bartel, Whitehead Institute/MIT 7. Todd Golub, Dana-Farber Cancer Institute, HMS*, Broad Institute 8. Bill Hahn, Dana-Farber Cancer Institute, HMS 9. Ed Harlow/Josh LaBaer, Harvard Institute for Proteomics, HMS 10. Eric Lander, Broad Institute, Whitehead/MIT/Harvard Composition of TRC-I and TRC-II * HMS: Harvard Medical School
7 Vector Used by TRC/RNAi Core http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Functional_Genomics_and_RNAi/Product_Lines/shRNA_Library.html EcoRI (GAATTC) AgeI (ACCGGT)
TRC2 library goals More powerful library: – enriched for best OT KD shRNAs – even coverage of genome TRC2 library 3+ good shRNAs per gene High-performing existing clones New clones(test)
9 Materials Received from TRC shRNA constructs and knockdown information: TRC-ITRC-II Clone #Gene #KDClone #Gene #KD Human81,88816,02634,89346,4285,5165,040 Mouse77,70015,97632,18831,2024,3891,753 Control85129 Total159,58832,00267,08177,7599,9056,793 Pooled genome-wide shRNA plasmid DNAs and chips (human and mouse) for RNAi genome-wide screening.
10 67,000 TRC shRNAs targeting 12,200 genes (Jurkat data excluded) J. Grenier All Hairpins StatisticsPer Gene Statistics TRC shRNA performance stats: Sept.09
How reproducible are the data?: How consistent is the > 70% call? Trial 1 Trial 2 >70% KD? +– + 1358 (39%) 498 (14%) – 391 (11%) 1248 (36%) Total hairpins: 3495 Jen Grenier, Shuba Gopal Consistent ‘pass call’ 75% of the time. 33% of inconsistent calls are > 60% KD in the failed rep 50% of inconsistent calls are < 80% KD in the passed rep
13 National RNAi Core (Since June 2005) Housed in Genomics Research Center (GRC) and Institute of Molecular Biology (IMB) Connected to TRC-I and TRC-II Responsible for Services Technology R&D Collaborative research activities Welcome local and international collaborations
14 Aims of RNAi Core: Third Phase Objectives: Maximizing the utilization of the RNAi library in mammalian genetic screen
15 Routine Service Items Categories Categories Service Items Service Items Bacterial Clones Bacterial Clones shRNA construct shRNA construct VSV-G lentivirus VSV-G lentivirus Arrayed VSV-G pseudotyped lentivirus Arrayed VSV-G pseudotyped lentivirus Pooled VSV-G pseudotyped lentivirus Pooled VSV-G pseudotyped lentivirus Individual VSV-G pseudotyped lentivirus Individual VSV-G pseudotyped lentivirus Customized lentivirus Customized lentivirus HT&HCS Image Analysis HT&HCS Image Analysis Plasmid DNA Plasmid DNA (lentiviral transfer vector) Package plasmids Package plasmids shRNA cloning lentivector shRNA cloning lentivector Pol II/ gene expression lentivector * Pol II/ gene expression lentivector *
16 Analyses of Geographic Distribution and Distributed Items to the Users Total Users: 465 PIs Bacterium: 79% Virus: 6% HTC image analysis: 2% Lentiviralvector: 13% 2% 79% 6% 13% Total Users: 465 PIs 11% 58% 3% 20% 8% AcademiaSinica(AS): 11% N.Taiwan(ASnot included): 58% Middle Taiwan: 8% Southern Taiwan: 20% Eastern Taiwan: 3%
17 User Publication 25 5 4 0 2 4 6 8 10 12 14 16 18 20 <55~10>10 Impact Factor # of publication 6 16 0 2 4 6 8 10 12 14 16 18 20 2006~20072008~2009 Year # of publication 12 2009~2010 Please acknowledge the RNAi Core for RNAi reagents when your research is get published. Please refer to http://rnai.genmed.sinica.edu.tw/faq-detail.asp?sn=16 http://rnai.genmed.sinica.edu.tw/faq-detail.asp?sn=16 for example.
18 Items Plan to Be Served in the Future Genome-wide RNAi pooled screening Produce/ provide shRNA-expressing lentivirus defined by User (a 2D barcode shRNA plasmid DNAs library is being established) Enlarge the activity of RNAi library screening C5: perform microarray and data analysis C5: perform microarray and data analysis C6: provide pooled virus C6: provide pooled virus
19 Collaborative Research Project(s) workflow and regulations Current regulations: Tying with the workload of the Core ; Tying with the workload of the Core ; Sharing cost and labor ; Sharing cost and labor ; Yes No Reject or Revise Yes Submission Re-submission RNAi screen and data analysis Data arrangement and release Discuss on the Potential project Review by committee Case closed Notify user committee Future capacity Could accommodate more projects Could accommodate more projects if transform Core into National RNAi if transform Core into National RNAi Screening Center (NRSC). Screening Center (NRSC).
Issues Regarding Lentiviral Transfer Vectors and shRNA/siRNA- triggered Off-Target Speaker: 鄭金松, RNAi Core Manager 98/10/16
3’LTR pLKO_AS2 EMCV IRES2 5’LTR puro Maps of lentivirus-based cDNA- expressing transfer vectors CMVie p CAG p Ubiqui p hPGK p EF1 p eGFP pLKO_AS3w pLKO_AS6w pLKO_AS7w pLEX_TRC203 0.6 kb 1.7 kb 1.2 kb 0.5 kb 1.26 kb
PCR Amplification of DNA Fragments Sticky-End PCR Method PCR 5’-CTAGC 3’-GATCG 5’-C 3’-G -3’ -5’ -3’ -5’ & Purify and mix 2 PCR products in an equimolar ratio; then de-nature and re-nature 5’-CTAGC -3’ 3’-G -5’ (Effective annealing PCR product for ligation) Primer #1 5’-CTAGC 5’ # Primer #2 Primer #1 5’-CTAGC 5’ # Primer #2 Separated PCR tube #1 Separated PCR tube #2 # Both reactions use the same reverse primer for PCR amplification.
(CAGp) (Ubqp)(hPGKp) (EF1 p) Expression Stability of Lentivirus-based System: Effect of Promoters Cell: A549 Wen-Ya
How long of insert can be tolerated in HIV-1-derived transfer vector?
55 O C 59 O C 64 O C 66 O C 69 O C 20 cycles Template: I l of 1 M M 1 2 3 4 3 Cycles 5 Cycles 8 Cycles 10 Cycles Template: I l of 50 M Optimization of the PCR Conditions for PCR-Based shRNA Construction
500bp 300bp 100bp M M 1 2 3 4 63bp 1.PCR products (5 cycles) 2.PCR cleaning 3.BsmBI digestion (before purification) 4.Column purified products (after digestion) Preparation of PCR Products for shRNA Cloning
500bp 300bp 100bp M 1 2 3 4 5 6 7 8 9 10 11 M 2 5 6 3 cycles (PCR) + 3 cycles + 2 + 3 Synthesized oligos have different amplification efficiency
Digestion Pattern of LKO_TRC shRNA Vectors AgeI/EcoRI double digestion
Stem-Loop Structure of shRNA May Interfere DNA Sequencing Without resolution buffer: With resolution buffer:
3’ UTR hexamer frequency in human genome SCF: seed complementary frequency high(>3800), medium (z2500–2800), or low (<350) SCFs in the HeLa transcriptome Khvorova A. RNA (2008),14:853-861.
Microarray signatures of GAPDH- and PPIB-targeting siRNAs Same seed sequences in different target genes: GAPDH H15 sense: 5-GAAGUAUGACAACAGCCUC PPIB H17 sense: 5-CGACAGUCAAGACAGCCUG One nt shift in seed sequence: GAPDH M1 sense: 5- GGCUCACAACGG GAAGCUU GAPDH M8 sense: 5- GCUCACAACGGG AAGCUUG Seed region not static Khvorova A. RNA (2008),14:853-861.
Anderson E. M. et.al. RNA;2008;14:853-861 GAPDH PPIB Off-Target Numbers of GAPDH- and PPIB-targeting siRNAs
Seed sequence plays major role in off-target GAPDH high(>3800), medium (z2500–2800), or low (<350) SCFs in the HeLa transcriptome (z10 siRNAs for each group) Khvorova A. RNA (2008),14:853-861.
How are the TRC library shRNAs processed into short dsRNAs? Implications: hairpin design, off-target effects A C GGGTCGAGCTGGACGGCGACGTAC T G TTTTTCAGCTCGACCTGCCGCTGCATG Which strand goes into RISC? (Strand that goes into RISC is more stable/abundant) Where does DICER cut? polIII transcription start and stop; evidence for DROSHA processing? shRNA processing TRC: Jen Grenier, Andrew Grimson, Ozan Alkan 22 nts
22mer 18,2854% 5% 21mer 39,0959% 10% 20mer 6,7602% 2% 23mer 45,61010% 11% 22mer 205,24946% 51% 21mer 40,4449%10% 23mer23,2635%6% r 4Ts r5Ts GG 23mer 5,2171% 11% 22mer 32,2797% 67% 21mer 8,0292% 17% 20mer 1,029<1%2% GG21merSenseStrandSeqncC G21merAntisenseStrandSTTTTT A T C G Length#reads% shRNA% strand r e e 3Ts 5Ts 4Ts GG e 3Ts m 4Ts } 17% 72% (5) (3) (4) Small RNA sequencing: all 26 shRNAs Done by Solexa sequencer
Configuration of siRNA Dual-Luciferase Reporter System : Polyadenylation signal sequence
List of All Possible Seed Complement Frequency Hexamer as an Example Common feature of SCF: Sequence with CGCG.