siRNA Screening to Identify Novel Modulators of Aβ Homeostasis

Manage webcast
Paul D. Kassner, Ph.D.
Alzheimer’s disease (AD) is the most common form of dementia and is an incurable, degenerative and terminal disease that is generally diagnosed in people over 65. The original amyloid hypothesis postulated that amyloid beta (Aβ) deposits are the fundamental cause of the disease. Aβ is derived from cleavage of amyloid precursor protein (APP) resulting in various peptide lengths (38-43 amino acids). Although the role of amyloidosis in AD has evolved over the years, aberrant production of Aβ peptides and their aggregation is still believed to play a key pathological role in AD, and has continuously been the focus of AD research.

This seminar will review how an Amgen laboratory identified novel modulators of Aβ Homeostasis via high-throughput siRNA screening. It will review how this lab performed a primary screen on a human CNS-derived cell line amenable to siRNA transfection. It will describe the results of the initial screen and the counter screen performed to filter out genes involved in cell viability. The webinar will conclude by describing the proteins identified with putative novel roles in Aβ processing and the lab’s next steps in further evaluating the function of these proteins via organotypic cultures, RNAi studies, gene knockout studies, and pharmacological tools.
Jun 12 2012
59 mins
siRNA Screening to Identify Novel Modulators of Aβ Homeostasis
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  • Corning® HepatoCells: A Novel Cell-Based Model for In Vitro ADME/Tox Studies Sep 30 2015 4:00 pm UTC 60 mins
    Rongjun, Zuo, Ph.D.
    This workshop will introduce Corning® HepatoCells for ADME/Tox study. Derived from primary human hepatocytes, Corning HepatoCells are a renewable hepatocyte-like cell line and retain most of the physiological properties of their parental hepatocytes, such as mature hepatocyte-like morphology and induction response to prototypical inducers of CYP3A4, 1A2, and 2B6 similar to primary human hepatocytes. An overview of the characterization of Corning HepatoCells for ADME/Tox studies will be presented, along with using the model system for prediction of clinical CYP induction. The attendees will gain an understanding of the capability of Corning Hepatocyte-cells for drug ADME/Tox study.
  • Your In Vitro ADME CRO: New Assays for Transport, Enzyme Inhibition & Induction Recorded: Jul 29 2015 60 mins
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    Achieving your ADME/Tox testing goals requires experience, quality data, and proper alignment with regulatory guidance. Failure to meet these important requirements can put your drug discovery and pre-clinical goals at risk.
    This presentation will provide an informative overview of how you can advance and reach your pre-clinical drug discovery goals. It will discuss the importance of core contract research capabilities, including enzyme induction, enzyme inhibition, and transporter interaction. In addition, we will review new capabilities and opportunities including CYP induction and SLC transporter assay services – all designed to align with regulatory agency guidance documents.

    Speaker Bio:
    David Stresser is the Program Manager of Corning® Gentest℠ Contract Research Services at Corning Life Sciences since 2001, having held prior positions of Product Manager and Study Director since joining Corning in 1998. Prior to this, he was a post-doctoral associate in the laboratory of David Kupfer at the University of Massachusetts Medical School in Worcester, Massachusetts. He did his graduate work in the laboratory of David E. Williams at Oregon State University in Corvallis, Oregon receiving a Ph.D. in toxicology in 1994. Dr. Stresser has authored or co-authored 40 articles or book chapters in the field of drug metabolism and has been an invited speaker at various national and international meetings, pharmaceutical companies, and universities.
  • Authentication of Cell Lines for Pre-Clinical Cancer Research Recorded: Jun 22 2015 58 mins
    Yvonne A. Reid, Ph.D.
    Join us on June 22nd for a special Corning-sponsored webinar presented by ATCC®.

    Abstract:
    Animal cell lines are important in vitro systems and tools for scientists in diverse disciplines such as basic cell biology, genetic mapping, gene expression and gene therapy. Cell line authentication and characterization are crucial in these activities, yet they are underappreciated by most research scientists. Over the years numerous cell lines have been shown to be misidentified due, in part, to poor techniques and inadequate verification of cell line authenticity. Technological advances have given rise to improved capabilities. Cell line authentication now requires a comprehensive strategy that employs several complementary technologies for systematic testing for morphology, microbial contaminations, cellular identity/cross-contamination as well as functionality. The validity of conclusions drawn from research data is dependent on consistent and unequivocal verification of cell line identity and function. It is estimated that the financial loss incurred by poorly characterized or misidentified cell lines is in the millions of dollars. An overview of the current technologies used to authenticate cell lines will be presented.

    Speaker Bio:
    Dr. Yvonne A. Reid joined ATCC in 1980 and during the mid-1980’s her research focused on the use of DNA hypervariable probes for the intraspecies identification of cell lines. The evolution of this work has led to the implementation of routine screening of all human cell lines by STR analysis. She co-chaired the ATCC SDO committee on the Development Consensus Standard on the Authentication of Human Cell Lines: Standardization of STR profiling. Dr. Reid has more than 30 years of experience in cell biology, immunology and molecular biology. As Collection Scientist for the Cell Biology Program for over 10 years, she was responsible for acquisition of new animal cell lines and hybridomas into the Cell Biology General Collection.
  • An Introduction To Cell Culture Recorded: Apr 30 2015 58 mins
    Mark Rothenberg, Ph.D.
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    Topics to be discussed include:
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    • The requirements needed to set up a cell culture laboratory
    • Challenges when performing mammalian cell culture and how to overcome them

    About our Presenter:
    Dr. Mark Rothenberg graduated from Emory University with his Ph.D. in Cell and Developmental Biology. Over the past 25 years, Mark has held positions in both academia and industry where he has developed an expertise in the areas of assay development and cell culture. He currently holds the position of Manager Scientific Training and Education with Corning Life Sciences.
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    Abstract:
    There is a great interest in application of human mesenchymal stem cells (hMSCs) in cell therapy and tissue engineering due to their self-renewal, multi-lineage differentiation, immunomodulation, and trophic potential. One of the challenges faced in the clinical application of hMSCs is the need for efficient expansion of these cells in vitro without altering their capacity. Serum-free mammalian cell culture media, in particular, require optimization of the expansion protocols. Even subtle changes in routine handling can have a significant impact on the cells’ potential.

    This seminar will cover the variables that can influence the desired regenerative and differentiation properties including medium selection, vessel surface treatment, impact of the cell source, and seeding density. We will also discuss how users can select the correct conditions for optimized growth and functionality.

    Speaker Biography:
    Brian Posey is a Product Development Manager for cell culture media at Corning Life Sciences. Brian has over 10 years experience in cell biology and industrial scale cGMP manufacturing of both liquid and powder cell culture media. Since joining Corning in 2012, Brian has lead numerous innovative technology projects for the media business ranging from customer technology transfer for production scale-up to developing new serum-free media for industrial and stem cell lines.
  • A Novel “Thaw and Go” Cell-based Model to Study SLC Transporters Recorded: Feb 24 2015 59 mins
    Na Li, Ph.D.
    Abstract:
    As a key determinant of drug pharmacokinetics, transporter mediated drug-drug interaction has garnered significant attention from the pharmaceutical industry and regulatory authorities. Corning offers a comprehensive list of tools to support drug transporter studies and recently introduced Corning® TransportoCells™ products to support in vitro assessment of drug interaction with SLC transporters. This new model provides a convenient “thaw and go” cell-based model with robust activity and consistent performance. In this webinar, we will provide an overview of Corning TransportoCells products along with applications for in vitro-to-in vivo correlation. Validation data will also be presented for the newly available TransportoCells products, including OATP1A2, OATP2B1, PEPT1, PEPT2, and NTCP.

    Presenter Biography:
    Dr. Na Li received her B.S degree in Biology from Fudan University, Shanghai, China, and her Ph.D. in Pharmacology from Dartmouth Medical School, Hanover, NH. Her major research focus is on drug transporters, including interspecies differences in hepatobiliary transporters, transporter quantification, and in vitro-to-in vivo extrapolation of drug pharmacokinetics. At Corning, Dr. Li contributes her expertise in in vitro drug transporter technology and its application in drug ADME.
  • 3D Tissue Models for Analyzing Dermatologic and Respiratory Systems Recorded: Jan 28 2015 51 mins
    Yukari Tokuyama, Ph.D.
    Join us on January 28th for a special Corning-sponsored webinar presented by ATCC®.

    Abstract:
    The significance of 3D tissue modeling opens up new possibilities for the study of complex physiological processes in vitro. Advances in cell isolation, media development, substrates, and growth surfaces are leading to culture environments that provide better biological and functional properties than traditional 2D cell culture. These models may provide a more predictive analysis and result in a more streamlined process of drug discovery and development. In this webinar, we will discuss recent developments in 3D modeling using ATCC primary and hTERT immortalized cells with specialized Corning® permeable support culture systems in dermatologic and respiratory studies.

    Presenter Biography:
    Dr. Yukari Tokuyama is a Field Application Scientist at ATCC. Prior to this role, she led the Stem Cell Product Development group and focused on products for human induced pluripotent stem cells and lineage specific differentiation. She earned her Ph.D. in Cell and Molecular Biology from the College of Medicine at the University of Cincinnati, where she studied the mechanism of genomic instability in cancer. She completed her post-doctoral training at the Oregon Health & Science University, Oregon National Primate Research Center, with a research focus on human and non-human primate stem cell biology.
  • Factors to Consider When Choosing Cell-based Assays for Use with 3D Cultures Recorded: Dec 16 2014 60 mins
    Terry Riss, Ph.D.
    Join us for a special Corning sponsored webinar presented by Promega Corporation.

    Cells cultured in 3D model systems often acquire relatively large in vivo-like structures compared to the thickness of a 2D monolayer of cells grown on standard plastic plates. Multicellular 3D culture systems containing more than one cell type and exhibiting formation of a complex extracellular matrix represent a more physiologically relevant environment, yet provide a challenge for assay chemistries originally designed for measuring events from monolayers of cells. There is an unmet need for guidelines for design and verification of convenient and effective assays useful for larger 3D microtissues. Critical factors to consider for each model system and cell type include effective penetration of detection reagents and/or complete lysis of microtissue structures using combinations of detergent and physical disruption. We will present the approach used to verify performance of a bioluminescent ATP detection assay for measuring cell viability, a caspase assay for detecting apoptosis, and cell stress reporter assays to detect mechanisms leading to cytotoxicity. Recommendations for factors to consider when verifying performance of cell health assays on 3D culture models will be presented.

    Speaker Bio:

    Dr. Terry Riss started the Cell Biology program at Promega Corporation in 1990 and has since held several R&D and Project Management positions. Dr. Riss managed development of cell viability, cytotoxicity, apoptosis, and protease assay systems and also led efforts to identify and promote multiplexing of cell-based assays to determine the mechanism of cell death. Dr. Riss now serves as Senior Product Specialist, Cell Health involved in outreach educational training activities including validating assay systems applied to 3D cell culture models.
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    Mark Rothenberg, Ph.D.
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    •The importance of choosing the correct dissociation method
    •Troubleshooting techniques for culturing healthier primary cells.
    •Other topics to be discussed include:
    -Choosing the optimal cell culture surface
    -Selecting the optimal cell culture media 
    -Choosing the correct vessel for scaling-up
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    Paula M. Flaherty
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    We will discuss a portfolio of Corning products designed to investigate specific stages of angiogenesis through standardized and quantitative in vitro cell-based assays.

    •HUVEC-2 Endothelial Cells—Widely studied endothelial cells that have been pre-screened for responsiveness to VEGF, a prototypic stimulator of angiogenesis
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    Biography:

    Paula Flaherty is a Technology Manager at Corning Life Sciences. Her team develops strategy and products focused on the modulation of in vitro cell behavior using extracellular matrix, media, vessel design, and growth factors. Prior to joining Corning Life Sciences, Paula studied retinal degeneration at the Berman-Gund Laboratory, Harvard Medical School in Boston, MA. She received her bachelor’s degree in Microbiology from the State University of New York and is an In Vitro Cell Biology Fellow, W. Alton Jones Cell Science Center in Lake Placid, NY.
  • Tips and Tricks to Help Develop and Run Microplate-based Assays Recorded: Sep 25 2014 59 mins
    Mark Rothenberg, Ph.D.
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    Critical parameters for achieving success with these assays include understanding the assay dynamics, the instrumentation involved in reading the assay, and, in the case of cell-based assays, the environment in which the cells are grown impacts cellular physiology. Factors such as microplate geometry, density, surfaces, and instrumentation all play important roles in the success of the assay.
    Learn how to:
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    •Choose the correct tip for your liquid handling needs
    •Determine the correct instrumentation and settings to run your assay
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    Dr. Katherine Strathearn
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    This webinar will provide a simple guide and review of novel cell culture vessels and surface technologies that have enabled researchers to improve suspension and adherent mammalian cell culture scale-up. Additionally, this webinar will cover certain parameters to consider when scaling-up cells. Selecting the correct vessel and cell culture conditions will increase throughput without increasing laboratory space, incubator space, or time.
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    Fang Tian, Ph.D. & David Randle, PhD
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    Label-free dynamic mass redistribution (DMR) responses in whole cells provide phenotypic activity profiles which are readily amenable to evaluation of compound activity and pharmacology. DMR responses obtained using the EGFR cell panel showed that Epic Technology can be utilized to evaluate receptor responsiveness to ligands and successfully predict drug response. Furthermore, label-free phenotypic responses can provide profiles of cellular signaling pathways downstream of receptor activation that may identify alternative targets for drug screening in the cell panel. In summary, combining Epic Technology and the EGFR genetic alternation panel offers convenient tools to screen for ligands or biologics that directly target or affect EGFR receptor biology.
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    David M. Stresser, Ph.D.
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  • Applications of Corning® FluoroBlok™ Permeable Supports Recorded: Jun 25 2014 57 mins
    Jeffrey Partridge
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    An improved version of this technology was developed, Corning FluoroBlok. As cells migrate or invade through a fluorescence blocking membrane, they are detected using a bottom-reading microplate reader or inverted fluorescence microscope. Cells remaining in the upper chamber of the insert are shielded from detection, allowing for quantitation of cell numbers in this homogeneous assay system.
    Recommended uses of the FluoroBlok system as well as recent improvements will be reviewed in this seminar.
  • Corning® Matrigel® Matrix: Material Properties, Applications and Proper Usage Recorded: May 22 2014 61 mins
    Shabana Islam, PhD
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    Deepa Saxena
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    We demonstrate expansion and functionality of several clinically relevant cell types including hPSCs, mesenchymal stem cells (MSCs), human keratinocytes (HKN), endothelial colony forming cells (ECFCs), NPCs and cell lines for biomanufacturing (CHO and Vero) on these surfaces. Corning Synthemax and ECM Mimetic cultureware are versatile surfaces compatible with multiple media for culture of various cell types providing a ready to use alternative to ECM protein coating where animal-free and defined conditions are desirable.
  • Corning TransportoCells™, A Novel “Thaw and Go” Cell-based Transporter Model Recorded: Mar 31 2014 54 mins
    Na Li
    SLC transporter-mediated drug–drug interactions (DDI) can significantly impact the pharmacokinetics and safety profiles of drugs. The regulatory agencies (FDA/EMA) recent guidance documents recommend testing six SLC transporters for potential DDI: OATP1B1, OATP1B3, OAT1, OAT3, OCT1 and OCT2. The 2013 drug transporter white paper published by the International Transporter Consortium (ITC) identified additional drug transporters relevant to drug development, including the Multidrug and Toxin Extrusion SLC transporters: MATE1 and MATE2-K. The webinar will introduce a novel cell-based SLC transporter model system - the recently launched “Corning™ TransportoCells™” - for studying regulatory agency recommended SLC transporters. The new system provides a convenient “thaw and go”, high performing mammalian cell model which supports regulatory agency recommendations for evaluating transporter mediated drug-drug interactions in vitro. In this webinar, we will provide an overview of the product validation and applications for the TransportoCells™ transporter model system. Validation data will also be presented for the newly available MATE1 and MATE2-K Corning™ TransportoCells™.
  • Materials and Systems for Optimizing 3D Cell Culture Environments Recorded: Feb 18 2014 64 mins
    Marshall Kosovsky, Ph.D.
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    Lesley A. Mathews Griner and Madhu Lal-Nag
    Cancer stem cells (CSCs), commonly referred to as tumor initiating cells (TICs), are thought to be the origin of replicating malignant cells that remain after the primary tumor is removed or eradicated. While the contribution of TICs to cancers of hematopoietic origin is well established, there is a growing body of evidence that supports the presence of TICs within solid tumors. This heterogeneity contributes to the intrinsic resistance of solid tumors to chemotherapeutics and eventually leads to therapeutic failure and patient death. Historically, screening for oncology directed compounds has been performed in 2-dimensional monolayer cultures which fail to replicate the complex architecture and microenvironment of tumors in vivo. To address this need using prototype manufactured 384-well round bottom clear black plates coated with hydrogel (commonly referred to as ultra-low attachment or ULA plates) we have developed a method to generate single cell type spheroids to accommodate their use in high throughput screening. These cultures were systematically compared to spheres grown in conventional plates and comparative imaging analyses were carried out to assess sphere formation. In addition, using the same plates, we have developed multicellular tumor spheroids (MCTS) composed of various cell types cultured in the same well. Differences in the cellular compositions of these spheroids in response to drugs can be quantified using high content imagers such as the WiScan from Idea-Bio and the High Throughput Flow Cytometer from IntelliCyt. These heterotypic, differentially labeled spheroids are a valuable asset in the generation of cell-based HTS assays capable of identifying molecules that selectively kill TICs and the cells comprising their microenvironment.
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  • Title: siRNA Screening to Identify Novel Modulators of Aβ Homeostasis
  • Live at: Jun 12 2012 4:00 pm
  • Presented by: Paul D. Kassner, Ph.D.
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