Horizon Discovery Ltd. COMPLIMENTARY
Tuesday, May 20, 2014 • 9:00am-12:00pm
Genome Editing Technologies and Applications
9:00am Welcome and Introduction
Arumugham Raghunathan, Ph.D., Head, Business Development, Cell Engineering & Cell Line Products, Horizon Discovery Ltd.
9:10 Targeted Genome and Epigenome Editing Using Engineered CRISPR and TALE Technologies
J. Keith Joung, M.D., Ph.D., Associate Chief of Pathology for Research; Associate Professor of Pathology, The Jim and Ann Orr MGH Research Scholar, Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital
Targeted genome and epigenome editing technologies have recently emerged as important tools for biomedical research and as potential reagents for therapies of gene-based diseases. In this talk, I will present our recent work on the clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nuclease platform for introducing targeted genome sequence alterations, including discussion about the latest specificity improvements developed by our group. I will also describe the creation and validation of new technologies for modifying specific epigenomic marks on histones and DNA that can be used to induce targeted alterations in endogenous human gene expression. Taken together, these methodologies provide transformative tools for understanding human biology and offer promising pathways forward for developing therapies based on targeted alterations of gene sequence and expression.
9:45 X-MAN Cell Lines – Enabling Translational Research
Chris Lowe, Ph.D., Director, R&D, Cell Line Engineering, Horizon Discovery
Technological advances continue to improve the affordability of whole-genome sequencing and drive the recent successes in human genetics, identifying genes responsible for Mendelian diseases and unraveling the mutations that predispose individuals to common complex diseases. However, identifying the associated mutations is only the first step in the therapeutic pathway. Understanding the involvement of a mutation in a disease or therapeutic pathway remains a challenge and has been hampered by the lack of suitable in vitro tools.
We have used rAAV‐mediated homologous recombination, a proprietary part of Horizon’s GENESISTM platform (which consists of rAAV, ZFN and CRISPR), to generate suites of isogenic cell lines, carrying specific endogenous mutations in genes such as KRAS, EGFR and PIK3CA, as well as endogenous reporters utilizing NanoLuc luciferase, a small enzyme engineered for optimal performance as a luminescent reporter, to investigate the roles of specific genes and mutations in response to therapeutic agents and demonstrate their utility in functional genomics and high-throughput screening.
10:20 Coffee Break and Networking
11:25 Novel Tools for Cell-Based Screening With Mixed Populations of Isogenic Wild-Type and Mutant Cell Populations
Ranjit S. Bindra, M.D., Ph.D., Assistant Professor, Departments of Therapeutic Radiology & Experimental Pathology, Yale School of Medicine
Cell-based screening is now a common approach to identify novel compounds and genes which regulate key biologic processes in cells. Live cell growth tracking is an especially useful tool for synthetic lethal screens, although current approaches are limited by the requirement for cell lysis, fixation and/or highly specialized imaging techniques. We recently developed a novel system to fluorescently label cell lines for use in screening assays. High expression levels of many fluorescent proteins without nuclear localization can be toxic in cells, and it can adversely affect the ability of automated cell identification programs to discriminate individual cells. To address these two potential issues, we engineered fusion fluorescent proteins which contain modified FK506- and rapamycin-binding protein (FKBP12) destabilizing domains (dd) on their N-termini, and nuclear localization signals (NLSs) on their C-termini. The FKBP12 dd is unstable in the absence of high-affinity ligands, such as rapamycin and a biologically inert derivative, Shield1. The addition of Shield1 blocks the destabilizing effect of the N-terminal domain dd. Thus, fluorescent protein expression can be induced at specific times by the addition of ligand. Fluorescence is localized to the nucleus by the NLS, which facilitates the identification of individual cells using imaging algorithms. We created fusion proteins for blue, yellow, and red fluorescent proteins (referred to as ddBFPnls, ddYFPnls and ddRFPnls, respectively).We chose to modify these specific fluorescent proteins because they have minimally overlapping fluorescence excitation and emission spectra. This particular feature makes them amenable for use in combination to identify and track multiple unique cell populations. We confirmed that multiple cell lines stably expressing ddBFPnls, ddYFPnls and ddRFPnls could be identified and counted in 384- and 96-well microplates, at a range of cell densities and timepoints, using several different imaging platforms. In addition, mixed populations of isogenic cell lines harboring key mutations were obtained from Horizon Discovery and tested with our fluorescent marking system. These fluorescent marking tools will be useful for researchers interested in cell-based screens, and they likely can be used for simultaneous cell tracking of multiple unique populations in vivo.
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