Nadine , Heimhofer and Mueller, Matthias (2018) OPTIMISING A CELLULAR SYSTEM FOR MONITORING THE CALCIUM DYNAMICS IN THE SARCOPLASMIC RETICULUM. Bachelor thesis.

Abstract

The sarcoplasmic reticulum Ca2+ ATPase (SERCA) fulfils an important role in the functioning of myotubes – cells which make up the skeletal muscles. Calcium ions (Ca2+) are stored in the sarcoplasmic reticulum (SR) and released into the cytosol when needed for muscle contraction. To be ready for the next contraction, the Ca2+ needs to be pumped back into the SR immediately, which is done by the SERCA. If the SERCA is impaired, it can lead to various diseases such as brody disease.
GCaMP is a genetically encoded calcium indicator (GECI) used to observe intracellular Ca2+ levels. By modifying it, it can be expressed in the SR. Induced pluripotent stem cells (iPSC) were transfected with the gene for the modified GCaMP, called GCaMPer, using the PiggyBac (PB) method, in the hopes of creating a massive overexpression. Previous experiments have shown that the fluorescence signal is dependent on the amount of GCaMP. The iPSC used also contain an inducible MyoD gene, letting them differentiate into myotubes.
These transfected cells were characterised with different methods. Analysis with flow cytometry shows that their fluorescence is 10 times higher than that of the previous experiment’s clones. This increase in fluorescence corresponds to the Western Blot analysis, where a massive overrepresentation in comparison to the old clones was also found. An immunostaining shows a clear overlap between the SR marker and the GCaMPer, which demonstrates the specific expression in the SR. Finally, in the functional drug screening system (FDSS), a drop in fluorescence can be observed when the cells get stimulated to release their Ca2+ from the SR into the cytosol, proving the functionality of the GCaMPer. The screening window of these clones clearly lies above the background, and can hence be used to find substances with the ability to modify the Ca2+ flux in the SR.

Item Type:	Article
Date Deposited:	02 Oct 2018 00:45
Last Modified:	02 Oct 2018 00:45
URI:	https://oak.novartis.com/id/eprint/37092