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Title: Identification of mesenchymal genes involved in prostate organogenesis by LongSAGE analysis
Author: Orr, Brigid
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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The LongSAGE technique was used to generate a comprehensive transcriptional profile of the neonatal rat prostate rudiment. LongSAGE is an open-ended and unbiased gene-profiling method. Two libraries were made from tissues in the female UGS; one library was prepared from the whole female prostate rudiment and the other library was prepared from the inductive mesenchyme (VMP) isolated from within the rudiment. Several essential factors known to be involved in prostate development were identified in the libraries, including low abundance cDNAs such as AR and Fgf10. This suggested that the approach had sufficient sensitivity to identify key mesenchymal factors. The two libraries were compared, highlighting genes that were VMP-enriched. Candidate mesenchymal transcripts were selected from the libraries, either by statistical analysis of the library comparison or an intuitive approach. The expression and abundance of candidate transcripts were quantified by qRT-PCR in the male and female neonatal UGS. Subsequently, candidate transcripts were verified as VMP-enriched and were quantified during prostate development by Northern blot analysis. The protein distributions of selected candidates were localised within the neonatal rat UGS by Immunocytochemistry, and the effect of testosterone treatment on the protein distribution was studied. To test the function of one of the candidate on prostate growth and development, recombinant protein was added to prostate rudiments grown in vitro. Finally, candidate transcripts were investigated in human prostate cancer associated fibroblast cells (CAFs) and normal prostate fibroblast cells (NPFs) by RT-PCR and Northern blot analysis. SAGE analysis, qRT-PCR and Northern blot analysis identified six candidate transcripts as VMP-enriched; Dlk1, Notch2, Nell2, MMP2 and MMP14. The transcript expressed of each candidate was most abundant in the developing rat prostate during the perinatal period. Proteins for Dlk1, Ptn and Notch2 were localised to mesenchymal cells of the neonatal VMP and ventral prostate (VP). Ptn expression was also associated with the basement membrane and cell-surface of the epithelial duct cells of the VP. Treatment of VP organs with recombinant DLK1 in vitro increased the organ size and epithelial branching. Also, PTN, NOTCH2, MMP2 and MMP14 transcript expression was observed in CAFs and NPFs. PTN and PTN and NOTCH2 showed a decrease in CAFs compared to NPFs suggesting a tumour-suppressive role.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available