eagle-i University of PennsylvaniaUniversity of Pennsylvania
See it in Search

Molecular Screening Facility (Wistar)

Directors: Hills, Robert, Ph.D.; Lieberman, Paul M., Ph.D.

Summary:

The Wistar Molecular Screening Facility is a shared resource facility accessible to Wistar and non-Wistar scientists. The mission of the facility is to enable investigators to 1) apply cutting edge technology and unique resources to discover molecular, genetic, and small molecule compounds suitable to further study the functions of poorly understood proteins, signaling pathways, and cells in complex biological processes relevant to human physiology and disease; 2) foster collaborations; and 3) fulfill the long-term translational goal of the Wistar Cancer Center of merging basic mechanisms of cancer biology with disease-relevant themes of early-phase drug discovery and new target identification.

The Shared Resource operates on a fee-for-service basis, providing expertise in bridging automated technology with the development of innovative assays for high-throughput chemical and functional genomic screens. The laboratory strives to possess the flexibility to accommodate diverse biological systems and a variety of investigator-developed assay types. While service is the primary role of the laboratory, the staff will also develop and implement new technology as needed to fulfill the needs of its users. Education and training is also part of the laboratory's mission, as trainees apply high-throughput screening experiments to their investigations. The combinations of these activities will provide scientists opportunities to develop new innovative basic and translational research, preliminary data for hypothesis driven research grant applications, and public-private partnerships.

The Wistar Molecular Screening Facility was developed with support from the Commonwealth of Pennsylvania Department of Community and Economic Development Keystone Innovation Zone initiative, The F. M. Kirby Foundation, The CLAWS Foundation, The Florence & Daniel Green Foundation, The McClean Contributionship, From The Heart Foundation, the Noreen O’Neill Foundation for Melanoma Research, NIH shared instrumentation grants, and an NCI Cancer Center Support Grant.

Affiliations:

People:

Resources:

Instruments

  • Biacore T200 ( Surface plasmon resonance instrument )

    "The Biacore T200 (GE Healthcare) utilizes the Surface Plasmon Resonance (SPR) technology to study biomolecular binding events on a chip surface. This technology has the advantage of being able to work with biomolecules in their native forms, without the need of introduction of label or tag (referred to ‘label-free’). The basic principle involves immobilization of a ligand on a sensor chip followed by continuous delivery of an analyte by a microfluidic system. The SPR-based technology continuously monitors the changes of mass deposition on the detector surface, and allows sensitive and reliable characterization of biomolecular interactions. Any protein, DNA, RNA, lipid, carbohydrate, polysaccharide, cell, virus, drug, drug like molecule (organic or inorganic) can be used as the ligand or analyte. Since the detection system is based on measuring total mass on the sensor chip surface neither ligand nor analyte has to be tagged. The SPR technology can provide quantitative data on:
       • Specificity: How specific is the binding between two molecules?
       • Concentration: How much of a given molecule is present and active?
       • Kinetics: What is the rate of association and dissociation?
       • Affinity: How strong is the binding?"

  • Biotek ELx405 microplate washer ( Microplate washer )

    "The ELx405 is an automated plate washer with a 96 channel head that can be used with 96- and 384-well plates. This plate washer also offers a sonicating wash bath for the pins to help minimize clogging and increase the cleaning capabilities. The Elx405 offers the flexibility of integration with the BioStak plate stacker, as necessary."

  • Biotek Microflo plate dispenser ( Microplate dispenser )

    "The Biotek Microflo uses a peristaltic pump mechanism to dispense liquids in as many as 8 channels, with each nozzle fed by a separate feeding tube. The entire tubing assembly is removable and can be autoclaved when sterility during dispensing is essential. The Microflo can be programmed to fill specific plate columns as desired by the user, and is capable of dispensing 2.0 to 2000 µL in increments of 1 µL. The priming/dead volume for this automated dispenser is about 6-10mL. In addition the Microflo can be integrated with Biostak plate stacker unit that can accommodate up to 30 plates."

  • Biotek uFill plate dispenser ( Microplate dispenser )

    "This is a syringe-based, automated plate dispenser with a 16-channel manifold with a volume range of 5uL to 1500uL for 384-well plates and 10-3000uL for 96-well plates. The syringe and tubing assembly is removable and can be autoclaved when sterility during dispensing is essential. The priming volume for this liquid dispenser is about 13-20mL. The uFill can also be integrated with Biostak plate stacker unit that can accommodate up to 30 plates."

  • Perkin Elmer Janus 96/384 Modular Dispensing Tool ( Liquid handler )

    "The laboratory is equipped with a Perkin Elmer Janus 96/384 Modular Dispensing Tool (MDT), automated liquid pipetting workstation for compound/reagent transfer and library management. The Janus MDT is equipped with a 384 well head for transferring volumes from 0.5 ul to 25µl with disposable tips and a 96 well head with capabilities of transferring volumes upto 235 ul. The MDT workstation is integrated with a plate stack unit with capacity for up to 50 microplates."

  • Perkin Elmer Janus Verispan 8 automated workstation ( Liquid handler )

    "The laboratory is also equipped with a Perkin Elmer Janus Verispan 8 channel independent pipetting workstation for use in reformatting libraries (e.g. dilution series, Hit-Picking) and inserting controls on assay plates. The Verispan 8-tip is designed for use with fixed washable tips, disposable tips in 20 ul, 200 ul, and 1 ml sizes, and disposable filter tips in 25 ul and 175 ul sizes, or both."

  • Perkin Elmer Operetta ( Fluorescence microscope )

    "The Perkin Elmer Operetta is an inverted epifluoresence microscope that utilizes a laser auto-focus option to automate acquisition of high-content images in up to 4 channels (three fluorescent plus brightfield) from 96- and 384-well microtiter plates or slides. It is equipped with a xenon arc lamp excitation source, a motorized objective wheel with bar-coded, interchangeable objectives (2X, 4X, 10X, 20X, 40X), an 8 position excitation filter wheel with matched dichroics, an 8 position emission filter wheel with interchangeable, bar coded filters for maximum detection of fluorescent labels, and a 1.3 megapixel, 14-bit Peltier cooled CCD camera. The Operetta uses Harmony, which includes a seamlessly integrated database for management of images, experimental information, data analysis, and metadata information. This provides the capability for convenient queries using a sortable data tree and cellular image analysis software to select a standard image analysis routine or develop custom analysis sequences to fit a project’s specific needs. Data can be stored locally on a 3.0 TB drive for short periods of time. An additional 10.0 TB network accessible RAID server pre-configured to immediately handle storage, management, and analysis of high-content data using the Columbus web-based interface is available in cooperation with the Bioinformatics Resource and the Center for Systems and Computational Biology. Columbus provides database access to an unlimited number of authorized users, which enables them to use Acapella and develop custom analysis sequences that cannot be achieved using standard analysis algorithms."

  • PerkinElmer EnVision Xcite Multilabel plate reader ( Microplate reader )

    "The laboratory has a PerkinElmer EnVision Xcite Multilabel plate reader with a 40-plate stacking carousel. In addition, the facility has access to a Perkin Elmer Top Count and Wallac Victor 2 ."

    "The EnVision Xcite plate reader is capable of standard multi-mode detection, including:

    1. Absorbance
    2. Fluorescence Intensity (FI)
    3. Luminescence
    4. Ultra Luminescence
    5. Time-Resolved Fluorescence
    6. Fluorescence Polarization (FP)
    7. AlphaScreen (Amplified Luminescence Proximity Homogeneous Assay)

    A wide selection of filters and dichroic mirrors make most dyes and dye-pairs accessible to the reader. Certain filters and mirrors are designed for fluorescence polarization (FP) assays."

  • Tissue culture suite ( Environmental chamber )

    "The Wistar screening facility has a tissue culture suite approved for work with BSL2 level pathogens. It includes:

    • Two 6ft. tissue culture hoods, which can house rapid dispense plate fillers (e.g. μFill) to dispense tissue culture cells into assay plates.

    • 4 CO2 regulated tissue culture incubators.

    • A benchtop centrifuge fitted with microtiter plate holders and an inverted phase-contrast microscope for use while passaging and preparing cells for assays.

    The BSL2 classification of this facility enables utilization of retroviruses (e.g. lentivirus) produced from cDNA and shRNA libraries."

Reagents

  • ChemDiv Discovery Chemistry Set ( Chemical library )

    "The Discovery Chemistry (DC) Set consists of 30,000 compounds from ChemDiv’s diverse drug-like small molecules."

  • ChemDiv Target Specific Chemistry Set ( Chemical library )

    "The Target Specific Chemistry (TS) Set consists of 20,000 compounds of which 8,000 compounds target GPCR, 5,000-kinases, 3,000-Ion Channels, 3,000-NHR, and 1,000-proteases."

  • Lankenau Chemical Genomics Center library ( Chemical library )

    "The LCGC library set is composed of ~120,000 unique compounds that pass all Lipinski and drug reactivity filters typically used in pharma to qualify drug-like libraries for high throughput screening (HTS)."

  • Maybridge "HitFinder" Library ( Chemical library )

    "The HitFinder™ Collection comprises 14,400 premier compounds representing the drug-like diversity of the Maybridge Screening Collection, using a clustering algorithm based on standard Daylight Fingerprints and Tanimoto similarity. All screening compounds fit Lipinski guidelines for "Drug-likeness" (logP <=5, H-bond acceptors <=10, H-bond donors <=5, Molecular Weight <= 500), and all have purity greater than 90%. Compounds have been selected to be non-reactive, ensuring fewer false positives and higher quality results."

  • Maybridge RO3 Fragment Library ( Chemical library )

    "The Maybridge RO3 Fragment Library is composed of 1500 rule of three compliant (MW<300 Da, cLogP <3, H-bond donors/acceptors<3) chemically diverse fragments with assured solubility."

  • Molecular Targeted Cancer Therapies collection ( Chemical library )

    "A collection of ~100 molecular targeted cancer therapeutics, representing a broad range of targets (e.g. kinases, epigenetics, chaperones, etc)."

  • Natural Products Discovery Institute extracts ( Natural product extracts chemical library )

    "A set of Natural Product extracts prepared from a variety of microbes and plants. The samples represent a spectrum of moderate-polarity and polar solvent preparations from liquid fungal fermentations, solid-substrate fungal fermentations, liquid actinomycete fermentations, and a wide variety of plant species selected to cover a maximum number of genera of higher plants."

  • NIH Clinical Collection ( Known bioactives collection chemical library )

    "The NIH Clinical Collection (NCC) is a plated array of approximately 450 small molecules that have a history of use in human clinical trials. The collection was assembled by the National Institutes of Health (NIH) through the Molecular Roadmap Initiative as part of its mission to enable the use of compound screens in biomedical research."

  • SelleckChem Bioactive Compound Library ( Known bioactives collection chemical library )

    "A unique collection of ~1000 bioactive chemical compounds, including FDA approved compounds, annotated inhibitors natural products, and chemotherapeutic agents. Bioactivity and safety confirmed by preclinical research and clinical trials."

  • Spectrum Collection ( Chemical library )

    "This is a collection of 2000 compounds (25 plates, 80 compounds/plate). About half of the collection are known bioactive agents, permitting the evaluation of hundreds of marketed drugs and biochemical standards. The other half of the collection are pure natural products and their derivatives. The collection includes simple and complex oxygen heterocycles, alkaloids, sequiterpenes, diterpenes, pentercyclic triterpenes, sterols, and many other diverse representatives."

  • The RNAi Consortium (TRC) Hs1.0 shRNA library ( shRNA construct library )

    "The Wistar Institute has purchased bacterial glycerol stocks of the complete (human and mouse) TRC shRNA library (Moffat, J. et al Cell, (2006) 124:1283-1298). The human library (TRC-Hs1.0) targets 15,000 annotated human genes and consists of 80,700 precloned constructs. The mouse library (TRC-Mm1.0) targets 15,000 annotated mouse genes and consists of 76,800 precloned constructs. The hairpin sequences, a 21-base stem and a 6-base loop, are each cloned into the pLKO.1 vector, which allows production of replication-incompetent lentiviral particles, transient or stable expression of the shRNA, and antibiotic (puromycin) selection of transfected or infected cells. On average, each gene is targeted by ~5 constructs. The shRNA sequences for a given target are distributed throughout the cDNA sequence of a target, including a shRNA clone targeting the 3'UTR for use in phenotypic rescue studies using cDNA expression constructs."

  • The RNAi Consortium (TRC) Mm 1.0 shRNA library ( shRNA construct library )

    "The Wistar Institute has purchased bacterial glycerol stocks of the complete (human and mouse) TRC shRNA library (Moffat, J. et al Cell, (2006) 124:1283-1298). The human library (TRC-Hs1.0) targets 15,000 annotated human genes and consists of 80,700 precloned constructs. The mouse library (TRC-Mm1.0) targets 15,000 annotated mouse genes and consists of 76,800 precloned constructs. The hairpin sequences, a 21-base stem and a 6-base loop, are each cloned into the pLKO.1 vector, which allows production of replication-incompetent lentiviral particles, transient or stable expression of the shRNA, and antibiotic (puromycin) selection of transfected or infected cells. On average, each gene is targeted by ~5 constructs. The shRNA sequences for a given target are distributed throughout the cDNA sequence of a target, including a shRNA clone targeting the 3'UTR for use in phenotypic rescue studies using cDNA expression constructs."

Services

  • Biological assay development for screening experiments ( Analysis service )

    "The overarching goal for Assay Development focuses on the timely delivery of robust and well validated biological assays ready for implementation in screening experiments. Users are encouraged to contact laboratory staff as early as possible and remain actively engaged throughout the entire process. The laboratory staff provide:

    • Expertise in cell biology, molecular biology, enzymology, biochemistry, pharmacology, and microbiology.
    • Facilities for propagation of bacteria, yeast, insect cells, mammalian cells, and viruses (i.e. baculovirus, retrovirus).
    • Expertise in expression and purification of recombinant proteins.
    • Equipment and expertise for maintenance, quality control and molecular characterization of normal and engineered cells for cell-based assays under standard biosafety containment up to and including BSL-2.
    • Experience with a variety of biochemical, cellular, immunological, and image based assays using an array of equipment compatible with bench scale or HTS scale experiments.

    Guidelines to developing a miniaturized bioassay for High-throughput screening

    The process of assay development includes the identification of assay types for screening and determination of structure-activity-relationships, development of assay reagents, optimization of assay parameters for signal intensity, signal window, and precision, adaptation to automation, scalability, and quantitative assessment of the assay's fitness for screening. Assay parameters to optimize assay's include sensitivity to enable identification of compounds with low-potentcy, precision of biological response between wells and plates, accuracy of positive and negative control compounds with known pharmacology towards the intended target, and economic feasibility."

  • Focused shRNA gene sets ( Material production service )

    "The Resource will format clones of focused gene sets (i.e. user defined, GO annotated gene family, etc.) in new 96 well microtiter plates for growth and preparation of plasmid DNA."

  • High-throughput-screening service ( Material analysis service )

    "Pilot screening

    After establishing an optimized assay protocol that meets acceptable criteria in a replicate plate test, a pilot screen of 2,000 to 3,000 compounds from a reference library with a high-degree of pharmacophore content is recommended. The purpose of the pilot screen is to assess the behavior of the assay conditions and automation procedures you have optimized during assay development under actual screening conditions. In addition, this pilot screen will be used to establish criteria for ‘hit’ conditions and selection. At this point, an assay protocol must be submitted for inclusion in our screening database.

    Production screens

    The facility has the capability to process upto ~100 microtiter plates per day depending on the assay type and number of steps. Facility staff will work with investigators/users to determine screen logistics (time considerations, libraries, amounts/volumes of reagents, etc). For newly developed assays, it is recommended that the assay conditions be tested in a pilot-scale screen (see above). For previously developed assays, demonstration of acceptable criteria in a single day replicate plate procedure will be sufficient to proceed with a production screen. When performing a production screen, it is preferable to process as many plates as possible during usage of the equipment and laboratory staff time. We strongly suggest screening all compounds in duplicate to enhance interpretation of data points.

    To schedule any screen, contact David C. Schultz. Do not make arrangements with staff of the facility. Please plan to schedule your screen well in advance of the date you would like to start your experiment to eliminate scheduling conflicts and to ensure that the libraries are properly formatted.

    Only screening facility personnel are permitted to handle library stock plates, and thus they perform all compound transfers from library plates into assay plates. Screening facility personnel will not be responsible for conducting any other parts of a screen unless agreed upon prior to the initiation of the screen, and will be charged as assisted use.

    The laboratory conducting the screen is responsible for purchasing all plasticware to be used for the screen, this includes pipette tips, plates, and any other materials specific to your screen (reagent troughs, plate seals, tissue culture reagents, assay reagents, labels for barcoding etc.). The screening laboratory has also negotiated some bulk discounting on plates and pipette tips for the liquid handling unit, and therefore, investigators can purchase these items from the screening facility at cost."

  • Individual shRNA clone distribution ( Material production service )

    "Cancer Center members can request shRNA clones from the TRC library. Clones can be ordered through a web-based system accessible through the Wistar Intranet."

  • Library management service ( Support service )

    "Laboratory staff is responsible for the collection, handling, and formatting of libraries for screening applications. In addition, the laboratory staff handles all library transfer procedures, including pinning compounds to assay plates and “hit” picking."

  • Molecular Screening Facility equipment access ( Access service )

    "Users must be trained by facility staff prior to using the instrumentation in an unassisted mode."

  • Molecular Screening Facility training ( Training service )

    "Laboratory personnel will provide users with training in the use of the walk-up equipment (e.g. bulk reagent dispensers, plate washers, and plate readers). Users must be trained by facility staff prior to using the instrumentation in an unassisted mode. To schedule a training session, please make an appointment with David C. Schultz prior to training on the instrumentation in the Facility. All training time on equipment is charged as assisted use."

  • Post-screening studies ( Support service )

    "The laboratory staff will assist users with cherry-picking requests of candidate ‘hits’ from primary screens, preparation of plates with serial dilutions for IC50/Ki measurements, and development of orthogonal assays and counter-screens to confirm the accuracy and rank order potency of identified compounds."

  • Tissue culture suite access ( Access service )

    "The Wistar screening facility has a tissue culture suite approved for work with BSL2 level pathogens. It includes:

    • A 6ft. tissue culture hood, which can house rapid dispense plate fillers (e.g. uFill) to dispense tissue culture cells into assay plates.
    • 2 CO2 regulated tissue culture incubators.
    • A benchtop centrifuge fitted with microtiter plate holders and an inverted phase-contrast microscope for use while passaging and preparing cells for assays.

    The BSL2 classification of this facility enables utilization of retroviruses (e.g. lentivirus) produced from cDNA and shRNA libraries."

  • Whole genome pooled library distribution ( Material production service )

    "The Resource has prepared pools of plasmid DNA for clones from the TRC shRNA library by species and produced concentrated viral vector (³5x108 TU/ml) for infection of mammalian cell cultures. Each plasmid DNA/viral vector pool consists of ~9600 constructs with the whole genome of each species (human and mouse) covered by 12 pools, respectively. The pools are not organized by GO annotated gene families. Each viral shRNA vector pool and control vector (e.g. empty, non-targeting shRNA) will be provided as 2 x 10ul frozen aliquots in PBS that have been verified to be at least 5 x 108 TU/ml, as determined using a p24 assay."


Web Links:

Last updated: 2016-08-02T17:29:50.802-04:00

Copyright © 2016 by the President and Fellows of Harvard College
The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016