Development of an In Vitro Model of Mitochondrial DNA Copy Number Depletion via Stable Inducible Expression of D1135A Mutant DNA Polymerase Gamma
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.