Using CRISPR to Model MCT1 Deficiency in Pluripotent Stem Cells.
2024
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Using CRISPR to Model MCT1 Deficiency in Pluripotent Stem Cells.
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2024
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Monocarboxylate transporter 1 (MCT1) plays a key role in transporting monocarboxylates such as lactate, pyruvate, and ketone bodies across the neurovascular unit/blood brain barrier (NVU/BBB). Human MCT1 is a highly conserved 500 amino acid protein embedded in the plasma membrane which contains twelve transmembrane segments with its amino and carboxyl termini in the cytoplasm (Figure). It acts as a facilitative carrier that transports a monocarboxylate and H + in equimolar amounts down a concentration gradient. Some carboxylate drugs may enter the brain via MCT1 and because of its role in metabolism, it has become the target for transport inhibitors (α-cyano-4-hydroxycinnamate, MD-1, AZD3965). Despite the important function of MCT1 in moving metabolically relevant substrates, there have been reports of mutations in the SLC16A1 gene, which codes for MCT1, and render the protein product dysfunctional. Patients with these mutations present with metabolic ketoacidosis that sometimes include seizures, vomiting, and mild to moderate developmental delay. There are currently no in vitro models of MCT1 deficiency in human cells. We hypothesized that creating a cell line of human stem cells in which the SLC16A1 gene is altered would be useful for characterizing the role of MCT1 in the metabolism of cells of the NVU. We therefore used CRISPR/Cas9 editing of human induced pluripotent stem cells (iPSCs). We were able to isolate a heterozygous KO cell line, and were further able to use the isolated line to attempt to create a full/homozygous knockout. The characterization of the resulting cell lines of this study will provide insight into how this pathology may affect the NVU, as well as provide a model system to further investigate this genetic disease.
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University of Minnesota M.S. thesis. 2024. Major: Chemistry. Advisor: Lester Drewes. 1 computer file (PDF); 46 pages.
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Reutzel, Bryan. (2024). Using CRISPR to Model MCT1 Deficiency in Pluripotent Stem Cells.. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/262859.
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