Yale HTCB Executive Summary
Outline
•! Mission Statement and Center Refocusing •! Timelines •! Personnel •! Resources •! Projects •! Challenges and Solutions •! Summary and Future Directions
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Mission Statement
•! The goal of the Yale Center for High Throughput Cell Biology is to provide the expertise, technology, and resources needed to advance the fields of molecular and cellular biology. Our current focus on RNAi screening underscores our commitment to the principle that probing the details of cell signaling by modulating gene expression will elucidate the mechanisms for life, disease, and death.
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Refocusing Center Efforts
•! "The Center will develop and provide assays and related services for RNAi, cDNA, Chemical Library and other high throughput screens that can be used to probe basic cellular mechanisms and, in due course, the function of human disease genes."
•! "The goal of the Yale Center for High Throughput Cell Biology is to provide the expertise, technology, and resources needed to advance the fields of molecular and cellular biology. Our current focus on RNAi screening underscores our commitment to the principle that probing the details of cell signaling by modulating gene expression will elucidate the mechanisms for life, disease, and death."
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Timelines
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May-08 June-09
Milestone April 1st:
Technological infrastruture
Milestone June 30th:
10 screens completed @10,000 genes per
screen
Timeline: Team-building
Lars Branden Director
Adrian Poffenberger Informatics
Marie-Aude Guie Michael Richo Phil Williams
Michael Wyler Assay Development / Implementation / Imaging
Deborah Smith Anthony Raffo Yan Song
Ashima Bhan Leena Kuruvilla
Susanne Hasse
Steven Berman Operations
Lori Ortoleva-Donnelly HTS
Jason Ignatius
Trisha D’Errico Administration
Laurie Tomei
January-09
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May-08 January-09 June-09
Milestone April 1st:
Technological infrastruture
Milestone June 30th:
10 screens completed @10,000 genes per
screen
Ordering Cell
Culture Equipment
Assemble Cell Culture work area
Issues with computers, campus mail, FedEx, shuttle, Corporate Express, Accuship, stock room purchases,
p-card
Renovations of HTCB space
Scope of work to be done Project Budget $300,000 – Project
Cost $260,000
Scheduled work orders for services still required
to get HTCB up and running
Purchasing process, invoicing, ISP, CDA and Contract Template
Timeline: Renovation and Yale Integration
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May-08 January-09 June-09
PLATE SEAL REMOVAL SYSTEM #1 and 2
STORAGE SYSTEM N3300 EXPRESS
APPLICATION SERVER ORACLE DATABASE SERVER
INCUBATOR HERACELL CO2 #1 and 2
Opera
Biorad Imaging system
#2 Enclosure
#3 Enclosure
CELLULAR SCREENING WORKSTATION #2 and 3
Immunostaining WORKSTATION #4
Milestone April 1st:
Technological infrastruture
Milestone June 30th:
10 screens completed @10,000 genes per
screen
UltraVIEW
DISK DRIVE SUBSYSTEMS
FREEZER #1-6 FREEZER CONTROL RATE
NITROGEN FREEZER ROBOT #1
Timeline: Equipment
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Dimaio Genetic Requirements of Senescence
Corson / Crews Triptolide Assay
Sessa LDL Uptake
Chapin PC-1 tail trafficking
Shadel Mitochondria Dynamics
Pober TNF
Saltzman Glioma stem cells
Fernandez-Hernando Cholesterol Trafficking
Mader Matrix degeneration in breast cancer
May-08 June-09
Milestone April 1st:
Technological infrastruture
Milestone June 30th:
10 screens completed @10,000 genes per
screen
Timeline: Projects
Schepartz Cell-Penetrating Miniature
Protein
Pritchard Transcription factor K-D
Scott Small APP-BACE
Oyagen EVVVHR Quenched FRET
Scott Small VPS35
Sigma Antibodies and Assays
HTCB NFAT Adeno Assay
HTCB NF!B validation assay
HTCB AP1 Adeno Assay
January-09
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Personnel
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Organizational Chart
Resources
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Tecan Evo Freedom 200 Highly advanced, fully automated liquid and plate handling platforms capable of processing 1536-well plates. Our platforms can deliver liquid volumes from 10 nanoliters to 125 microliters and can process over 200,000 wells per day. Barcoded screening plates allow digital plate tracking and real-time data streaming to our enterprise-scale sample, plate, and results database.
Perkin-Elmer Opera High throughput imaging system for high content sub-cellular imaging and analysis of more than 70,000 samples/day in three colors.
Infinite 1000M Plate readers are integrated with the automation platforms to provide a broad spectrum of detection capabilities including fluorescence, luminescence, absorbance, and FRET.
Infrastructure
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Infrastructure •! Scalable hardware infrastructure housed within a modern industrial-scale data center. •! Databases and data processing applications primarily utilize IBM x3850, IBM x3650, and Mac Xserves in
addition to other systems. •! Computational projects are supported internally via our 24-CPU Linux cluster. For larger projects we utilize the
Yale High Performance Computing Cluster, which currently provides 1000 CPUs. •! Primary storage is managed by an IBM n3300 SAN device. Hard disk space is presently over 10TB and is
quickly scalable to 80 TB on existing controllers and equipment. •! Backups are performed on an IBM TS3200 tape device capable of archiving over 75 TB per batch, and backups
are managed using an IBM x336 server. Long-term archiving is performed on a Blu-Ray optical disc jukebox. •! Network infrastructure provides 1 Gb/s connectivity throughout the Yale West Campus facilities and 10 Gb/s
fiber to the Internet. •! Our primary scientific applications include ActivityBase suite (IDBS); Pipeline Pilot (Accelrys); MetaCore
(GeneGo); and Acapella (PerkinElmer) for high content image analysis and feature extraction. •! Fully validated industry-scale disaster recovery plan.
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Dharmacon siGenome Human siRNA Library
Total: 21,122 genes
Druggable Genome: 5906 genes
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Projects
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Projects
•! Free-screens for Yale Investigators
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Yale Free Screens
•! October 3, 2008 – Introductory email sent to Yale community along with request for proposals for free screens.
•! October 20, 2008 – Deadline for free-screen applications.
•! Response rate: 37 applications received for 10 free screens out of 9,562 email solicitations sent.
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Initial Screen Purpose: To find Binding Partners of PKD2 Involved in Triggering Cell Death.
Model System – Triptolide is toxic to Hela Cells. Biochemical studies demonstrated
that triptolide binds to PKD2, a calcium channel present in the plasma membrane.
Assay – Treat cells with triptolide and 24h later, measure cytoplasmic ATP levels.
Problem – Positive controls did not work as expected, i.e., siRNA to PKD2 and
caspase-3 did not block reduction in ATP at 24h.
Final Screen Purpose: Screen for targets related to triptolide toxicity.
Assay -- Treat cells with triptolide and use luminescent assay for caspase-3
activation at 18h.
Corson – Triptolide P.I., Craig Crews
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Projects
•! Clients beyond Yale University
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Oyagen Assay-240K Compound Screen Harold Smith P.I., Oyagen, Rochester, NY
Assay Description –! VIF, an HIV encoded
protein, forms a dimer that allows for infection by disrupting antiviral activity of a human enzyme, APOBEC
Assay Principle –! Transfect with EVV:VHR
and look for compounds that prevent VIF dimerization
–! Quenching assay EVV EVV:VHR
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Sigma-Aldrich Antibody Validation
0
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40
60
80
100
120
140
Membrane Nuclear Cytoplasm Perinuclear
1:50
1:100
1:200
1:400
1:800
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1:3200
1:6400
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Genetics of Transcription Factor Targets Jonathan Pritchard, Yoav Gilad at Univ of Chicago
HapMap Project: International Effort to
study patterns of human genetic variation.
Nigerian (Yoruba) European
Japanese
Chinese
Project: Target Transcription Factor genes with shRNA in a lentivirus Vector. Prepare total RNA for qPCR and Microarray.
Project Phase 1: One Cell line: B-lymphocytes (EBV transformed) (Yoruba)
50 Targets: 48 TF genes plus 2 Controls
5 shRNAs per gene In Triplicate
750 RNA
Isolations 24
Genetics of Transcription Factor Targets Jonathan Pritchard, Yoav Gilad at Univ of Chicago
HapMap Project: International Effort to
study patterns of human genetic variation.
Nigerian (Yoruba) European
Japanese
Chinese
Project: Target Transcription Factor genes with shRNA in a lentivirus Vector. Prepare total RNA for qPCR and Microarray.
Project Phase 2: Five Cell Lines
2000 Targets: 1998 TF genes plus 2 Controls
5 shRNAs per gene In Triplicate
150,000 RNA
Isolations 25
Projects
•! Informatics Projects
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Informatics Projects •! Protein Half-life prediction •! Gene Profiler •! siRNA off-target prediction •! Mouse/human siRNA target gene overlap •! Parallelized image analysis •! Secure web-based client data access •! Fully integrated image management
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Hypothetical diagram of molecular turnover time comparison
siRNA levels decline
from dilution and degradation
Protein level declines from
normal turnover with non-replacement due to silencing
Incubation Time
after transfection
Concen
tration
Optimum time window
for experimental measurement
Protein level
returns to normal
Transfection started
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NFkB Signaling Pathway
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Highest average interaction score
Shortest path length
Highest common gene count
Multiple algorithms
Assay Plate: hits in red
Gene Profiler
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