IDENTIFYING THE FUNCTIONS OF SOLUTE CARRIER PROTEINS

Abstract

Long-lived plasma cells (LLPCs) take up more 2-(7-Nitro-2,1,3-benzoxadiazol-4-yl)-D-glucosamine (2NBDG), a fluorescent glucose analog, than do short-lived plasma cells (SLPCs). However, the mechanism of 2NBDG uptake is independent of known glucose transporters and thus remains undefined. Identifying the 2NBDG transporter might help us understand differences in SLPC and LLPC biology. Here we outline an optimized clustered regularly interspaced short palindromic repeats (CRISPR )-Cas9 genome editing method for investigating the role of solute carrier (SLC) proteins, a broad family of transporters responsible for the movement of small and mostly hydrophilic metabolites across cellular membranes, in 2NBDG uptake. A gRNA plasmid based CRISPR system was inefficient at inducing individual SLC knockouts in 293T, even after optimization of transfection conditions. Sequencing confirmed that measured changes in cell viability and metabolite levels upon transfection were not attributable to SLC knockouts. In comparison to plasmid transfections, the ribonucleoprotein CRISPR system was more effective at inducing SLC knockouts in 293T cells for general metabolic and 2NBDG uptake analysis. As the specific target solutes and functions of many SLC family members have yet to be identified, establishment of an efficient and effective method to investigate these functions provides many avenues for metabolic discovery, including but not limited to furthering our understanding of plasma cell biology.