Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Pathology and Laboratory Medicine

Supervisor

Castellani, Christina A

Abstract

Mitochondria are responsible for several crucial cellular processes and contain their own DNA (mtDNA) that exists in several copies. Variation of mtDNA copy number (mtDNA-CN) alters energy metabolism and can modify the epigenome and transcriptome. We hypothesized that inducible expression of polymerase-deficient D1135A dominant-negative DNA polymerase gamma (DN-POLG) would result in mtDNA-CN depletion. Here, an in vitro model expressing D1135A POLG was created using the Flp-InTM T-RExTM-293 stable inducible expression system. Stable integration was confirmed with PCR amplification and Sanger sequencing of post-integration genomic sequences. D1135A POLG expression was confirmed with Western blot of the FLAG-tag antibody. Induction of D1135A POLG expression with tetracycline for 24 hours resulted in reproducible decreases in mtDNA-CN. This model will be used in the future by the Castellani Lab to interrogate the effects of mtDNA-CN depletion on the nuclear epigenome, transcriptome, and metabolome.

Summary for Lay Audience

Mitochondria are involved in many cell functions, including energy production. Mitochondria contain their own DNA (mtDNA) that exists in several copies per cell. Variation of mtDNA copy number (mtDNA-CN) alters energy production and has been shown to modify the nuclear epigenome, altering expression of nearby genes. mtDNA is replicated by DNA polymerase gamma (POLG). Different regions (domains) of the POLG protein are responsible for mtDNA editing (exonuclease domain) and replication (polymerase domain). Mutations in the polymerase domain have been shown to decrease mtDNA-CN in cell culture. We hypothesized that expression of polymerase-deficient D1135A POLG would result in mtDNA-CN depletion. A cell model expressing D1135A POLG was created using the Flp-InTM T-RExTM-293 cell line, which contains a Flp-recombinase target (FRT) site and allows the D1135A sequence to be incorporated into the genome via transfection with a plasmid also containing a FRT site. Stable integration of the gene of interest into the Flp-InTM T-RExTM-293 cell line was confirmed with PCR amplification and Sanger sequencing of DNA sequences unique to the cell line after integration. D1135A protein expression was confirmed with Western blot of the FLAG-tag antibody. D1135A POLG expression was induced with tetracycline treatment for 24 hours, resulting in reproducible decreases in mtDNA-CN. This model will be used in the future by the Castellani Lab to interrogate the effects of mtDNA-CN depletion on the nuclear epigenome, transcriptome, and metabolome.

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