Establishing a detection system for anti gene doping purposes

Nøhr, Morten Klitgaard

4. term

Medicine with Industrial Specialisation, Master

2013

2013-05-30

44 pages

Background Doping becomes more prevalent in the society and the sports world. A relatively new doping method has been added to the prohibited list issued from the World anti-doping agency, which is gene doping. The use of the preproenkephalin gene has the potential of becoming a doping candidate. The gene encodes two enkephalin proteins, that bind to the µ-, δ-, and κ-receptors which exert analgesic effects. It is therefore believed that the insertion of this gene into an athlete can induces higher pain tolerance, which will provide him/her with an unnatural benefit in sports. AIM To insert the preproenkephalin gene as a potential candidate for gene doping against pain by electroporation method and to establish a detection method in blood and muscle for inserted enkephalins using quantitative polymerase chain reaction. Method Plasmid encoding the genes for mouse preproenkephalin (PENK) and green fluorescent protein (GFP) was transfected into mouse muscles cells using electroporation. A control group was also electroporated, which received a saline solution instead of plasmid. The cells were extracted after 24 and 48 hours, which were then analysed using a qPCR assay. An in vitro experiment was performed to investigate if PENK plasmid could be efficiently expressed in C2C12 myoblast cells after transfection using a cationic polymer as transfection agent. This was visualised on confocal microscopy. Results The results showed that it is possible to insert and detect the PENK gene in the muscle. It was not possible to detect the gene in the blood after electroporation. The C2C12 transfection confirmed that the PENK gene could be efficiently expressed. Small clusters were visualised in the C2C12 cells indicating the PENK-GFP proteins are packed in vesicles. Conclusion This study indicates that it is indeed possible to use PENK as a gene doping candidate. Furthermore, it was impossible to detect this gene using qPCR at other sites then the actual transfected muscle. Thus, blood samples failed to detect the PENK gene, and as such making gene doping by electroporation ideal as a doping method. As such, it is imperative for anti doping purposes to find practically applicable techniques that would be able to detect this type of gene doping

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