Faculty
Schulich Medicine and Dentistry
Supervisor Name
Dr. Christina Castellani
Keywords
mitochondria, alpha-ketoglutarate, TFAM knockout, mtDNA-CN
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Description
The mitochondrion is a double membraned organelle possessing a distinct set of DNA (mtDNA). Recent studies point towards retrograde signaling in playing a critical role in disease whereby mitochondria are involved in translating environmental stimuli into epigenetic changes. More specifically, my project takes a special interest in how fluctuating availability of mitochondrial metabolites impact retrograde signaling. Alpha-ketoglutarate is one such metabolite as it activates demethylases TET and JMJD to increase DNA and histone demethylation.
TFAM is a nuclear gene involved in mtDNA replication and repair. Dr. Castellani had previously used CRISPR-cas9 to induce heterozygous TFAM KO cells which demonstrated an 18 fold reduction in mtDNA. HEK293T cells were first used to optimize a colorimetric a-KG assay kit’s standard curve and sample prep workflow. The kit was then applied to assay 3 TFAM KO and 3 TFAM NC lines in singleplex to produce preliminary results.
Acknowledgements
Thank you to my supervisor Dr. Christina Castellani and Castellani Lab members Amanda Morin, Phyo Win and Hasti Gholami for the immense support and guidance they provided me this summer. As well, thank you to the Western USRI program and the Schulich School of Medicine and Dentistry for this valuable experience.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License
Document Type
Video
Assaying alpha-ketoglutarate content in TFAM knockout cell lines - Ziwei Lin (Schulich Medicine and Dentistry)
The mitochondrion is a double membraned organelle possessing a distinct set of DNA (mtDNA). Recent studies point towards retrograde signaling in playing a critical role in disease whereby mitochondria are involved in translating environmental stimuli into epigenetic changes. More specifically, my project takes a special interest in how fluctuating availability of mitochondrial metabolites impact retrograde signaling. Alpha-ketoglutarate is one such metabolite as it activates demethylases TET and JMJD to increase DNA and histone demethylation.
TFAM is a nuclear gene involved in mtDNA replication and repair. Dr. Castellani had previously used CRISPR-cas9 to induce heterozygous TFAM KO cells which demonstrated an 18 fold reduction in mtDNA. HEK293T cells were first used to optimize a colorimetric a-KG assay kit’s standard curve and sample prep workflow. The kit was then applied to assay 3 TFAM KO and 3 TFAM NC lines in singleplex to produce preliminary results.