Mitochondrial dysfunctions, which underlie many systemic diseases in animals and humans, may arise from accumulation of mutations in the mitochondrial genome. PolG-alpha enzyme encoded by Polg gene is crucial for replication and repair of the mitochondrial genome. The aim of this study was to assess the possible role of Polg mutations in mitochondrial dysfunctions using in vitro and in vivo animal models. The experiments involved transgenic mice with inducible expression of Polg mutant variant; the methods included cell culture, real time PCR assay, fluorescence flow cytometry, and skeletal muscle functional tests. The results indicate that mouse embryonic fibroblasts (MEFs) expressing Polg pathogenic mutant variant have decreased mitochondrial membrane potential and increased expression of mitophagy markers compared with control cultures. Transgenic animals with systemic expression of the pathogenic variant develop mitochondrial dysfunction which significantly affects muscular performance. In addition, systemic expression of mutated Polg in transgenic animals significantly inhibits expression of TCR subunit α and CD3 coreceptor complex subunits δ and ε in total splenocyte populations and significantly affects cellularity of the thymus without altering its CD4/CD8 subpopulation ratio. Thus, inducible expression of mutated Polg in transgenic animals provides a relevant model for studying mitochondrial dysfunction and its treatment in vitro and in vivo.