Genome engineering and patient-derived pluripotent stem cell technology have dramatically changed our abilities to understand disease pathways. These two technologies are recent additions to our investigative armamentarium but have rapidly permeated all aspects of biomedical science, providing unprecedented power to discover how perturbations of components in specific pathways lead to disease. The Genome Engineering Core C is composed of 11 investigators and all of the necessary equipment, reagents and expertise required to perform the experiments needed to support the research goals of the 3 projects outlined in this PO1 renewal application entitled ?New therapies for liver fibrosis and hyperproliferation in alpha1-AT deficiency (ATD)?. Our facility is housed within the Washington University Genome Engineering and IPSC Center (GEiC) and was established and is overseen by the Department of Genetics. It was created to facilitate the implementation of these powerful new technologies in laboratories at Washington University. All projects proposed within this PO1 renewal application plan to extensively utilize the services provided by the Core. These experiments are largely aimed at the ongoing evaluation of modifying variants in ATD and their potential exploitation for development of new treatments for this disorder. The services to be utilized include the design, construction and validation of genome editing reagents (e.g. gRNAs, donor plasmids, and Cas9 derivatives). The Core also produces modified cell lines using genome editing technologies, including gene knockout, variant introduction, epitope tagging or gene replacement. These cell lines will be used to better understand the biology of ATD and to assess the impact of variants selected for their potential to modify disease progression and facilitate drug development. The Core will also design and generate materials needed for rapid production of animal models harboring selected variants. The Core produces iPSCs from both skin biopsies and the renal tubular epithelial cells present in urine samples. Genome engineering of iPSCs is now routinely performed in the Core to introduce new variants and to convert disease-associated mutant alleles back to wildtype or use as controls. Skin biopsies (fibroblasts) or urine (renal tubular epithelia) will be procured from ATD patients or controls and the cells will be reprogrammed to produce iPSC lines. Genetically modified patient-derived iPSCs will be generated using genome editing techniques. In addition, the personnel in the Core provide assistance for investigators in performing genome engineering, particularly in the development of new techniques and reagents, and for the maintenance and differentiation of iPSC lines.
The Genome Engineering Core C provides reagents, services, and instruction critical for success of the projects outlined in this application. Each of the projects uses genome engineering to produce modified cell lines or animals that will be essential for assessing the importance of modifier variants to be studied in these projects. The Core will be invaluable to the PO1 research teams in providing reagents as well as ensuring that new aspects of these technologies are disseminated to participating laboratories to help expedite their research.
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