Evaluating the ability of a transient transfection MitoTimer method to measure mitochondrial biogenesis in C2C12 cells

David Grandjean Gleerup

Studenteropgave: Speciale

Abstrakt

Mitochondrial dysfunction has been linked to several negative conditions, one of them being the age related loss of muscle function known as sarcopenia. The current methods of surveying mitochondrial turnover are mainly limited to indirect assessment of mitophagy and measurements of protein turnover. It would therefore be beneficial to discover new and more direct ways to assess aspects of mitochondrial turnover. Recently one such potential method has emerged, named MitoTimer. This method is based on the fusion between the Cytochrome C oxidase subunit VIII targeting sequence and the fluorescent protein named Timer. The result is a protein that changes its fluorescence from green to red at a fixed rate and accurately localises to mitochondria. This creates the possibility to assess both mitochondrial biogenesis and mitophagy in one experiment. In this thesis we investigated the accuracy of the MitoTimer method in an experimental setup using transient transfection, which would give the possibility to assess the mentioned functions without the need for establishment of a stable cell line. To assess the viability of a transient transfection, we transfected C2C12 cells with a plasmid containing the MitoTimer protein and then treated the cells with various compounds known for their ability to stimulate or inhibit biogenesis. To confirm the reliability of the results we then used the fluorescent stain known as MitoTracker to measure mitochondrial mass and a Seahorse flux analyzer XF24 to measure oxygen consumption rate to confirm biogenesis. It was found that a transient setup is likely not viable for MitoTimer as the results were unstable and at points self-contradictory. Furthermore, they generally did not match the results from neither MitoTracker nor Seahorse.

UddannelserMolekylærbiologi, (Bachelor/kandidatuddannelse) KandidatMedicinalbiologi, (Bachelor/kandidatuddannelse) Kandidat
SprogEngelsk
Udgivelsesdato2020
VejledereOle Vang

Emneord

  • mitochondria
  • MitoTimer