In force

Application of microarray technology for the detection of changes in gene expression after doping with recombinant hgh - part 2

Principal investigator
G. Gmeiner
Country
Austria
Institution
ARC Seinersdorf Research Labororatory GmbH
Year approved
2003
Status
Completed
Themes
Growth Hormone (GH)

Project description

Code: 03C01GG

The present project aims to introduce microarray technology as a new analytical tool into the field of doping control analysis to gain insight in specific effects of recombinant human growth hormone (hgh) on blood cells (leukocytes). Microarray technology will be used to search for changes in leukocyte gene expression which are directly related to the application of human growth hormone (hgh). Knowledge generated along this research project and the introduction of microarray technology into doping analysis should open novel approaches and strategies for future detection of doping substances. In the first phase of the project cell lines of specific leukocyte subsets (THP-1/monocyte, IM-9/T lymphocyte, H9/B lymphocyte), PBMCs (peripheral blood mononuclear cells) from healthy donors, respectively will be treated in vitro with hgh to identify candidate genes influenced by hghapplication. Whole genome cDNA microarrays as well as oligonucleotide microarrays including leukocyte-relevant genes will be used to characterize potential hgh-specific genes by comparing gene expression profiles of treated and untreated cells. To find additional hgh-candidate genes, suppression subtractive hybridization (SSH) technology will be applied on hgh-treated PBMCs and on those cultured leukocyte subsets which showed a response to hgh before during in vitro studies. After confirming SSH-genes to be differentially expressed in a series of microarray experiments, oligonucleotide probes will be designed for both SSH-genes and the candidate genes identified along gene expression profiling using whole genome/leukocyte relevant arrays. To establish a selective “hgh-chip” the newly designed oligonucleotide probes for the hghcandidate genes will be printed on a microarray. The “hgh-chip” will then be thoroughly evaluated by performing numerous gene expression experiments on hgh-treated and untreated leukocyte subsets, cultured PBMCs as well as PBMCs obtained from hgh-patients. During the proposed project new, nucleic acid-based analytical techniques will be used to gain insight into specific effects of hgh on gene expression of leukocytes. Experience and expertise gained along this project should be of great importance for novel strategies esp. in fields of doping analysis, where so far no satisfying analytical test exists.