Projects & Scientific Cores

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Core B: Mutagenesis and Cell Expression

Jen Pan, Ph.D.
Carlos Vanoye, Ph.D.

Our Center is studying a large number of previously uncharacterized epilepsy-associated genetic variants in human ion channels using a high-throughput strategy (Project 1). We will also 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 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) is responsible for these two broad activities.

To accomplish this work in an efficient, cost-effective, and rigorous manner, Core B exploits standardized high-throughput workflows for site-directed mutagenesis and heterologous cell transfection that unite innovative technologies with economies of scale. Service activities are 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.

Three core services are offered by Core B. First, to enable the large scale functional evaluation of ion channel variants proposed in Project 1, Core B is generating and validating hundreds of site-specific mutations in human NaV and KV channel cDNAs in vectors optimized for mammalian expression. Human brain NaV channels (NaV1.1, NaV1.2, NaV1.3, and NaV1.6) are notoriously difficult to handle because of intrinsic instabilities of the corresponding recombinant plasmids, but we recently discovered the molecular basis for this instability and have succeeded in constructing stable versions of these channel plasmids that are compatible with high-throughput mutagenesis. The primary work will be with human channel cDNAs, but select studies in Project 1 and 3 will require mutant constructs in mouse orthologs. Although it is widely assumed that human and mouse mutations behave similarly, we won't make this assumption when selecting variants for creation of new mouse models. An essential step prior to engineering new mutations in mice will be a demonstration that human and mouse mutations cause the same or highly similar functional consequences when expressed in the orthologous ion channels.

The second service line involves the generation of heterologous cell models. The primary platform proposed in Project 1 for functional evaluation of human variants is automated planar patch clamp recording. Typically, this platform requires cell lines stably expressing the protein of interest to ensure a high success rate, but substantial time and cost barriers would be incurred by analyzing hundreds of variants. To obviate a need to generate stable cell lines for all variants, Core B is exploiting a validated, high-efficiency cell electroporation method for transiently expressing channel variants in cultured cells that has proven to be useful for automated electrophysiology. This method is being used for transfection of common cell lines (CHO-K1, HEK293) and a novel neuronal cell line that we engineered to be devoid of endogenous NaV channels (ND7/No-Nav). A subset of variants for which pharmacological studies are planned will be expressed stably using inducible promoters. Core B is responsible for validating transfections, quality controlling stable cell lines, and cryopreservation and distribution of cells.

The third service provided by Core B will be design and construction of targeting vectors for introducing point mutations in the murine genome using the CRISPR/Cas9 system or similar genome editing technology. This service will directly support Project 3. All engineered plasmids, targeting constructs, and cell lines generated by Core B will be shared with the academic community.

Highlights of Core B:
• Established workflows for high throughput mutagenesis and high efficiency cell electroporation
• Availability of stabilized human neuronal NaV channel plasmids and novel cell resources
• Tight integration with functional evaluation projects