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Day Two:
Tuesday, November 16
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Functional Silencing with RNAi |
7:30-8:15 Technology Workshop (sponsorship available)
8:30 Chairperson's Remarks
Dr. Sumit Chanda, Group Leader, Genomics Institute of the Novartis Research Foundation
8:40 Combination of Fluorescent Protein and RNAi: Towards Mammalian Cellular Genetics
Dr. Shin-ichiro Kojima, Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University
Green fluorescent protein and its derivatives can serve as easy and reliable markers of gene silencing by RNA interference. Vectors co-expressing fluorescent markers along with hairpin RNA facilitated purification of knockdown cell populations and rescue of knockdown by introducing exogenous genes fused with a different color fluorescent protein. Therefore, a genuine genetic approach, which was afforded previously only by a few model organisms, is now readily feasible in mammalian cell culture. Design of experiments and application to cell biological analysis will be presented.
9:10 Genomic View of siRNA-mediated Gene Silencing with GFP Protein
Dr. Jen-Tsan Chi, Assistant Professor, Molecular Genetics & Microbiology, Duke University
To examine the gene expression change associated with triggering RNAi mechanisms in human cells, we have silenced the GFP protein expression by
siRNA-mediated gene silencing and assess the global gene expression changes with cDNA
microarrays. These results will be essential for large RNAi genetic screening and
RNAi-based therapeutic efforts.
9:40 Genome-scale Functional Profiling Through Image-based Analysis
Dr. Sumit Chanda
Cellular assays provide a powerful platform for the interrogation of gene function. Application of highly parallelized methods to examine cellular phenotypes can enable the study of gene activities at the level of the genome. Towards this end, we have assembled an arrayed collection of approximately 25,000 (full-length) cDNAs and 15,000
siRNAs, and developed high-throughput methodologies for parallel transduction of various cell-types. In conjunction with high speed microscopy platforms, we have executed cell-based assays to examine a diverse range of activities, including
nuclear-cytoplasmic translocation, proliferation and apoptosis, molecular aggregate formation, cell migration, and mitotic events. To date, application of functional profiling technology has led to the elucidation of hundreds of novel gene activities, and will likely play an integral role in the understanding of gene function on a global scale.
10:10 Coffee Break, Poster & Exhibit Viewing
10:40 Poster Teardown
10:50 Functional siRNA Screening of the Cell Cycle with a Dynamic Cellular GFP Sensor
Dr. Nick Thomas, Principal Scientist, GE Healthcare Bio-Sciences
We have used high-throughput sub-cellular imaging in concert with a dynamic GFP sensor to screen and profile the effects of siRNA knockdown of a panel of more than 100 genes on the cell cycle in a mammalian model system. The use of the GFP sensor coupled with image analysis allowed siRNA effects on cell number, morphology and cell cycle position to be correlated with data from complementary microarray and molecular assays of mRNA and protein levels in the same model system. Perturbation of a sensitive and dynamic phenotypic cellular assay via siRNA provides a powerful tool for functional analysis of the cell cycle.
11:20 mRNA Fusion Constructs Serve in a General Cell-based Assay to Profile Oligonucleotide Activity
Dr. Dieter Hüsken, Department Functional Genomics, Section Nucleic AcidsSciences, Novartis Institutes for BioMedical Research Basel
RNA interference mediates a process of sequence-specific posttranscriptional gene silencing by double-stranded RNA. Short interfering RNAs (siRNA) has recently emerged as a powerful genetic tool to aid in the analysis of gene function in mammalian cells and it is a key approach to the linking of genes to disease-linked substrates, pathways and pathologies. As with antisense technologies, the levels of silencing efficiencies are variable and to identify the most potent reagents siRNAs are best characterized before experimentation. The siRNA method comprises multiple molecular interactions: association with the transfection reagent and uptake, assembly and activation of the guide strand with the RNA-induced silencing complex, binding to the mRNA target structure, target cleavage and degradation. We have developed a reliable quantitative cellular based assay for the rapid and efficient identification of the most effective siRNA against any piece of mRNA sequence. Efficacy of siRNAs is monitored by their ability to inhibit a surrogate target, a reporter-cDNA mRNA fusion, with an easily quantified automated readout, fluorescent protein (eYFP).
11:50 Chairperson's Introduction
Dr. Richard Levenson
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Plenary
Presentation |
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Held in conjunction with In Vivo Molecular Imaging
12:00 In Vivo Imaging with Green Fluorescent Proteins
Dr. Robert Hoffman, President, Anticancer Inc.
In vivo imaging with green fluorescent proteins has begun a revolution in research on small animals. Processes that could not be previously visualized in a living animal can now be visualized down to the single-cell level with use of fluorescent proteins. Multiple processes can be simultaneously imaged using fluorescent proteins of different colors. Applications of
in vivo imaging with fluorescent proteins will allow the development of the new field of
in vivo cell biology that can be applied to all types of disease models.
12:30 Luncheon in the Exhibit Hall
1:45 Chairperson's Introduction
Dr. Robert Hoffman
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Plenary
Presentation |
Held in conjunction with In Vivo Molecular Imaging
1:50 Multispectral Imaging for Enhanced Fluorescence Detection
In Vivo
Dr. Richard Levenson, Director of Research and Development, Biomedical
Systems, CRI, Inc.
Fluorescence-based detection in vivo benefits from molecular specificity, multiplexing capability, extensibility from animal models to some clinical applications, a plethora of probes, targets and labeling strategies, and relatively low equipment cost. However, especially in the visible emission range (500 to 700 nm) it may be hampered by bright tissue autofluorescence that limits achievable signal-to-noise. Multispectral imaging, which captures images at multiple wavelengths, combined with algorithms that can
"unmix" signals, results in labeled targets appearing brightly and quantitatively displayed against a black, near-zero background. Using the CRI Maestro™ imaging system which can operate from 500 to 950 nm (emission wavelengths), we have demonstrated relatively deep detection of GFP signals from orthotopically implanted
GFP-labeled lung tumors, separation of GFP, RFP (red flouresent protein) and autofluorescence signals in transgenic mice, highlighting of vascular features thanks to a mixture of fluorescence and absorbance phenomena, and quantitative detection of RFP glial tumors through intact mouse skulls, among others. The technology is fast (under a minute for both acquisition and spectral unmixing processing), has high spatial resolution (can be used for zebrafish embryos as well as for imaging multiple mice), is reproducible, highly sensitive and easy to use. |
2:20 Close of Conference
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