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DEVELOPMENT OF A GENE-EXPRESSION ARRAY FOCUSING ON THE HYPOTHALAMUS-PITUITARY-THYROID AXIS IN XENOPUS LAEVIS
Citation:
Korte, J J., J E. Tietge, AND S J. Degitz. DEVELOPMENT OF A GENE-EXPRESSION ARRAY FOCUSING ON THE HYPOTHALAMUS-PITUITARY-THYROID AXIS IN XENOPUS LAEVIS. Presented at 3rd Indo-US Workshop on Mathematical Chemistry, UMD, Duluth, MN, August 2-7, 2003.
Description:
As recommended by the Endocrine Disruptor Screening and Testing Program Advisory Committee (EDSTAC), the USEPA has been developing a screening test capable of detecting effects of Endocrine Disrupting Chemicals (EDCs) on the hypothalamus-pituitary-thyroid (HPT) axis in Xenopus laevis. As part of this, we developed an oligonucleotide array to assess changes in expression of important thyroid-axis genes, allowing comparison of the results with morphometric and biochemical information. The method consists of hybridizing amplified biotinylated RNA from test organisms with membrane-bound synthetic DNA oligomers of 65 to 70 bases. The biotin label is detected by chemiluminescence and quantified. Currently, there are at least two different oligos for each of the 70 different different genes represented on the array, and each oligo is spotted twice. The system shows robust responses from housekeeping genes (actin, elongation factor 1-a, ribosomal protein L8, etc.), as well as strong responses from genes important to the HPT axis. The method has numerous attractive features: 1) very sensitive, surpassing radioactive methods 2) system does not require complete sequence information, useful in species such as Xenopus laevis where complete genomic information is not available 3) high flexibility, allowing genes to be added as new sequences become available 4) does not require robotics or other highly specialized/expensive equipment 5) fast, the results are available a few hours after the hybridization step. The array has been utilized on brain samples from stage 54 to 62 tadpoles to determine the pattern of gene expression in control animals during development. Future studies with known modulators of thyroid function will compare expression patterns to normal tadpoles to look for indications of altered gene expression. Ultimately, we hope to utilize the method to indicate if the thyroid axis has been impacted by potential EDCs.