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Title: Gene expression profiling in rat tissues by cDNA representational difference analysis
Author: Hu, C. T.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2004
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Gene profiling was performed using a PCR-based subtractive hybridisation method, called Representation Difference Analysis (RDA), and kidney tissue from congenic versus hypertensive controls for the SHRSPGla. The RDA method itself I have also refined using glycerol-enhanced mini-polyacrylamide gel electrophoresis to optimize the separation of differentially expressed RDA fragments. I have isolated 20 such fragments that are differentially expressed and have been able to positively identify them by BLASTA search against known sequences on Genbank. These include the cytochrome P450 4A3 gene, which has been well documented as differentially expressed between SHR and WKY kidneys, and the low affinity Na-dependent glucose transporter (SGLT2). The expression of SGLT2 mRNA has been shown to be kidney specific and I have shown that it is differentially expressed in the congenic strain of SP.WKYGla2a versus the parental SHRSPGla. Two novel genes have also been mapped onto our target chromosome 2 within the congenic interval by radiation hybrid (RH) mapping. Others have also been mapped onto chromosome 2 but they are also linked to other chromosomes, suggesting either a technical problem with PCR typing of the radiation hybrids, homologous gene products or most likely epistatic gene-gene effects. The CN3.1 fragment shows almost 100% homology to 4 EST rat clones (AA924217, BF282696, AI547398 and AA893369). Importantly, the first of these has been positionally cloned in silico to chromosome 2 on the Rat Genome Database. It has also been possible to align one important clone I pulled out to a supercontig on chromosome 2. Above all, it is intriguing that the original cDNA-RDA fragment has now been identified as the novel hypothetical protein LOC310133 in chromosome 2. Furthermore work is needed to further characterize the novel cDNA fragments I have identified as mapping to the region of the chromosome 2 QTL. I hypothesise that one of them may be the pressor gene for this rat QTL.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available