? Core B Our Center proposes to study a large number of previously uncharacterized epilepsy-associated genetic variants in human ion channels using a high-throughput strategy (Project 1). We also propose to generate novel mouse models of prototypical variants after we demonstrate that mutant mouse ion channels exhibit patterns of dysfunction similar to the corresponding human variant (Project 3). Both of these research endeavors will rely heavily upon the engineering of variants in recombinant ion channels and the generation of expression systems in heterologous cultured cells. In addition, Project 3 requires the design and construction of targeting vectors for generation of novel mouse models using genome editing technologies. The Mutagenesis and Cell Expression Core (Core B) will be responsible for these two activities. To accomplish this work in an efficient, cost-effective, and rigorous manner, Core B will exploit standardized high-throughput workflows for site-directed mutagenesis and heterologous cell transfection that unite innovative technologies with economies of scale. The specific variants to be generated will be determined by the Variant Prioritization and Curation Core (Core A). Service activities will be distributed between the Translational Neurobiology program at the Broad Institute of Harvard University and MIT and the Department of Pharmacology at Northwestern University to take advantage of advanced recombinant DNA and cell engineering expertise on these two campuses. Core B will provide the following services: 1) to engineer recombinant human ion channel plasmids and variants for functional evaluation; 2) to generate and distribute heterologous cells transiently or stably expressing human ion channel variants; and 3) to generate and heterologously express prototypical mouse ion channel variants for comparison with the corresponding human variants and to construct murine targeting vectors.
