Expression of SLC25A46 in Knockout Zebrafish

Hi! I'm Hank Ingham, a senior at Pine Crest High School in Fort Lauderdale, Florida; my topic is "Expression of SLC25A46 in Knockout Zebrafish." I worked on this project at the University of Miami's Hussman Institute for Human Genomics under the supervision of Elena Buglo.

Abstract

Charcot-Marie-Tooth disease (CMT) is a common inherited neurological disorder, affecting one in 2,500 people. The chief symptoms are loss of muscular tissue and touch sensation. SLC25A46 is a gene responsible for mitochondrial membrane fission; mutations in it can cause CMT, as well as optic atrophy and ataxia. Zebrafish are increasingly important model organisms in genetics because they reproduce quickly, are cost-effective, and have nervous systems and genomes similar to those of humans.

To examine the specific effect mutant SLCA46 has on an organism, the gene was knocked out in zebrafish with CRISPR/Cas9 gene editing. Edited fish had abnormally-shaped neurons when viewed under a microscope, but the F2 generation exhibited no phenotypic difference. Sequencing confirmed the DNA was edited, however. RT-qPCR showed no downregulation of mRNA production, as would be expected because of nonsense-mediated decay. To study protein expression, a plasmid with the mutant gene was transfected into a human cell line, and then analyzed through Western blot to check upregulation, and staining and microscopy to check localization. Both tests were inconclusive; if when repeated they show no difference, one possible explanation could be that a homologous gene was upregulated to compensate; RNA sequencing could be used to determine this.