Establishing a detection system for anti gene doping purposes
Author
Nøhr, Morten Klitgaard
Term
4. term
Publication year
2013
Submitted on
2013-05-31
Pages
44
Abstract
Baggrund: Gendoping er kommet på Verdens Antidopingagenturs (WADA) forbudsliste. Et muligt mål er preproenkefalin-genet (PENK), som koder for enkefaliner – stoffer der binder til opioidreceptorer og dæmper smerte. Tanken er, at indsættelse af PENK i en atlet kan øge smertetolerancen og give en uretfærdig fordel. Formål: At indsætte PENK som potentiel gendoping mod smerte via elektroporation (korte elektriske impulser, der hjælper DNA ind i celler) og at udvikle en metode til at påvise det i blod og muskel med kvantitativ polymerasekædereaktion (qPCR). Metode: Et plasmid med musens PENK og grønt fluorescerende protein (GFP) blev indført i muskel hos mus ved elektroporation. En kontrolgruppe fik saltvand med samme procedure. Muskelprøver blev taget efter 24 og 48 timer og analyseret med qPCR. Derudover blev dyrkede musemuskelceller (C2C12 myoblaster) transficeret med PENK-plasmid ved hjælp af en kationisk (positivt ladet) polymer, og udtrykket blev visualiseret med konfokalmikroskopi. Resultater: PENK-genet kunne indsættes og påvises i muskelvæv, men det kunne ikke påvises i blod efter elektroporation. I C2C12-cellerne blev PENK udtrykt effektivt. Små klynger blev set i cellerne, hvilket indikerer, at PENK-GFP-proteinerne pakkes i vesikler. Konklusion: Studiet viser, at PENK potentielt kan bruges som gendoping mod smerte. Samtidig blev genet kun påvist i det transficerede muskelvæv og ikke i blodprøver med qPCR, hvilket kan gøre denne fremgangsmåde svær at opdage med standard blodtests. Derfor er der et presserende behov for praktisk anvendelige metoder, der kan afsløre denne type gendoping.
Background: Gene doping has been added to the World Anti-Doping Agency’s (WADA) prohibited list. One candidate is the preproenkephalin gene (PENK), which encodes enkephalins—molecules that bind opioid receptors and reduce pain. The idea is that inserting PENK into an athlete could raise pain tolerance and confer an unfair advantage. Aim: To deliver PENK as a potential pain-related gene doping approach using electroporation (brief electrical pulses that help DNA enter cells) and to establish a detection method in blood and muscle using quantitative PCR (qPCR). Methods: A plasmid carrying mouse PENK and green fluorescent protein (GFP) was introduced into mouse muscle by electroporation. A control group received saline with the same procedure. Muscle samples were collected at 24 and 48 hours and analyzed by qPCR. In parallel, cultured mouse muscle cells (C2C12 myoblasts) were transfected with the PENK plasmid using a cationic polymer, and expression was visualized by confocal microscopy. Results: The PENK gene was successfully inserted and detected in muscle tissue but was not detectable in blood after electroporation. In C2C12 cells, PENK was efficiently expressed. Small clusters were observed, indicating PENK-GFP proteins were packaged in vesicles. Conclusion: This study indicates that PENK could serve as a gene doping candidate for pain modulation. It was detectable only at the transfected muscle site and not in blood by qPCR, suggesting this approach could evade standard blood testing. This underscores the urgent need for practical methods to detect this form of gene doping.
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