Journal of Sports Science and Medicine
Journal of Sports Science and Medicine
ISSN: 1303 - 2968   
Ios-APP Journal of Sports Science and Medicine
Androit-APP Journal of Sports Science and Medicine
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©Journal of Sports Science and Medicine (2012) 11, 294 - 303

Research article
Fundamental Study of Detection of Muscle Hypertrophy-Oriented Gene Doping by Myostatin Knock Down Using RNA Interference
Tohru Takemasa1, , Naohisa Yakushiji1, Dale Manjiro Kikuchi2, Custer Deocaris1, Widodo3, Masanao Machida1, Hidenori Kiyosawa1
Author Information
1 Health and Sport Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan
2 Biological Sciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
3 Biology Department, Faculty of Sciences, Brawijaya University, Jl. Veteran, Malang, Indonesia

Tohru Takemasa
‚úČ Physical Education, Health and Sport Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ten-noudai 1-1-1, Tsukuba, Ibaraki 305-8574, Japan
Email: takemasa@taiiku.tsukuba.ac.jp
Publish Date
Received: 14-10-2011
Accepted: 22-03-2012
Published (online): 01-06-2012
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ABSTRACT

To investigate the feasibility of developing a method for detection of gene doping in power-athletes, we devised an experimental model system. Myostatin is a potent negative regulator of skeletal muscle development and growth, and myostatin-knockout mice exhibit a double-muscle phenotype. To achieve knockdown, we constructed plasmids expressing short hairpin interfering RNAs (shRNAs) against myostatin. These shRNAs were transfected into C2C12 cultured cells or injected into the tibialis anterior (TA) muscle of adult mice. By performing in vitro and in vivo experiments, we found that some shRNAs effectively reduced the expression of myostatin, and that the TA muscle showed hypertrophy of up to 27.9%. Then, using real-time PCR, we tried to detect the shRNA plasmid in the serum or muscles of mice into which it had been injected. Although we were unable to detect the plasmid in serum samples, it was detectable in the treated muscle at least four weeks after induction. We were also able to detect the plasmid in muscle in the vicinity of the TA. This gene doping model system will be useful for further studies aimed at doping control.

Key words: In vitro and in vivo transformation, C2C12 cultured cell, tibialis anterior, real-time PCR, shRNA


           Key Points
  • Using a myostatin knockdown plasmid, we have succeeded in creating a model system for gene doping using mice that resulted in muscle hypertrophy greater than that reported previously.
  • We confirmed that there was a limit of gene doping detection using real-time PCR, either from serum or muscle smple.
  • This model experimental system can be utilized for examining indirect methods of gene doping detection such as immune responses to gene transfer or a profiling approach using DNA microarray.
 
 
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