einstein-montefiore cfar virology core director: dr. ganjam v. kalpana, ph.d
DESCRIPTION
Einstein-Montefiore CFAR Virology Core Director: Dr. Ganjam V. Kalpana, Ph.D. Overarching Goal. To provide CFAR Investigators with the appropriate support, training and access for the most relevant and cutting-edge approaches to rapidly and efficiently support their virology research programs. - PowerPoint PPT PresentationTRANSCRIPT
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Einstein-Montefiore CFAR
Virology Core
Director: Dr. Ganjam V. Kalpana, Ph.D.
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Overarching Goal
• To provide CFAR Investigators with the appropriate support, training and access for the most relevant and cutting-edge approaches to rapidly and efficiently support their virology research programs.
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Virology Core Aims• Providing the Infrastructure, reagents, assays and technologies to
enable basic and translational researchers to Investigate HIV Infection, pathogenesis and therapy;
• Performing cellular and molecular assays characterizing HIV-lnfection to support clinical and translational investigators engaged in patient-based studies of HIV/AIDS patients; and
• Providing training to investigators In a wide range of cellular and molecular assays used in the study of HIV pathogenesis.
• Maintaining a central repository of molecular clones of infectious HIV, viral isolates, reporter HIV clones and HIV reporter cells lines as well as HIV-based expression vectors.
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Virology Core Training ProgramsMeasurement of p24 antigen by ELISA Co-culture, quantification and sequence analysis of HIV
from PBMCs Determining the viral titers, infectious units (i.u.) and
m.o.i. of HIV using reporter cell linesReal-time PCR for quantification of HIV RNA and
cellular gene expressionApplication of advanced virological techniques to
analyze all stages of HIV-1 replication-including entry, reverse transcription, nuclear localization, integration, transcription, assembly and particle production.
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Virological Core Services• HIV culture and isolation • HIV p24 antigen capture assay • Repository of titered primary HIV isolates • HIV-1 neutralization assays • Quantitative HIV-1 DNA and RNA measurements• HIV and cellular RNA quantification by real-time qPCR• Determination of viral tropism. • Reporter virus entry assay. • HIV sequence analysis. • Patient sample processing and storage.• Immunosorting of control and HIV-infected cellular
subpopulations. • Measurement of serum HCV antibody and plasma HCV virus
levels.
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Molecular Biological Core Services• Routine
o Custom vector and plasmid design and construction o Provision and expression of reporter and full-length molecular
clones of viruses of many cladeso Site-directed mutagenesiso Large-scale expression of recombinant HIV, human or other
pathogen proteins
• Advanced o Lentiviral vector construction and preparation of virus
particles o RNA interference analysis o Genetic and biochemical systems to study protein-protein
interactions o Chromatin immunoprecipitation (ChIP) techniques o FRET-based BLam assay (beta-Lactamase based assay) o Heteroduplex mobility assay (HMA)
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• The core (in collaboration with ATRP program) purchased a new Applied Biosystem qPCR machine to facilitate the demand for use of this assay in studying HIV-1 infection.
• Core is developing methods to carry out microRNA analysis in HIV-1 infected samples.
• In collaboration with Epigenomics and Genomics Center, the core is planning to optimize the methods for RNA/microRNA-seq, ChIP-seq and Affymatrix techniques. Consultation for the developing and carrying out these techniques will be provided for CFAR users.
Expansion of Core Activities and Innovation
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Expansion of Core Activities and Innovation
M200spSample preperation
station
M2000rtRT-PCR machine
In collaboration with Bronx WHIS (PI: Dr. Anastos) and the Einstein’s Microbicide program (PI: Dr. Herold), CFAR has
purchased an Abbott m2000 machine
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Expansion of Core Activities and Innovation
• Abbot m2000 Provides automation for sample preparation and real time PCR.
• Ideal for using a variety of clinical samples and allows the use of bar coded tubes, creates an efficient workflow, minimizes errors and contamination.
• Barcoded Laboratory Tubes, Precision Pipetting-no manual mixing or manipulation, Open Mode-Flexible protocol for various sample types and volumes
• Efficient Sample Extraction--Flexible throughput options of 24 to 96 samples—allows isolation of nucleic acids, RNA or DNA from variety of samples (serum, plasma) based on the magnetic bead method.
• Samples are prepared automatically and loaded onto 96 well plates and sealed for transfer to PCR machine.
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Expansion of Core Activities and Innovation --- continued
• Abbot m2000 currently being used for HIV-1 viral load assays (range 40 to 10,000,000 copies/mL).
• Can be used for other clinical applications.Other viral loads—HCV, HBV
Detection of cellular transcripts-biomarkers, depending on the availability of the kit to use in the Abbott machine.
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Expansion of Core Activities and Innovation --- continued
Technical Innovations—Alpha Technology
AlphaLISA-instead of p24 ELISAVarious enzymatic- Kinase AssaysProtein-protein interactions
A reduced number of assay steps---no washing steps.
Automated---good for adopting to HTS Versatile—measure large biomolecules (up to 2000 kDa) or weak interactions (mM).
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Proposed Activities forNext Project Period
• The Core will expand its sequencing activities for molecular characterization of clinical isolates
• Expansion of virological support for WIHS studies
• Expansion of support to carry out epigenetic (methylation and ChIP assays) and microRNA analysis during HIV-1 pathogenesis
• Expand service to include support of protein production
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Accomplishments
• Over the past 5 years, we have contributed to data in 31 publications
• In the past year we have supported studies by 32 NlH-funded investigators.
• We have expanded the Core services In response to the needs of Investigators including developing highly cost-effective lab-developed assays.