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Title: The GABAA receptor δ subunit gene promoter : characterisation and use
Author: Roberts, Alexa Brett
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1998
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Viral vectors represent powerful tools in the rapidly developing field of gene therapy of the mammalian central nervous system. Interest in HSV-1 in particular as a vector relates primarily to its ability to establish long-term latent infections in neuronal cells. A goal of this research was the development of a safe HSV-1 vector that would deliver a reporter gene to target cells in the CNS and restrict its expression to a specific cell-type by the use of a neuronal promoter. A second goal was to add to the present limited understanding of neuron-specific promoter structure. The murine GABAA receptor delta subunit gene promoter was selected for use as a prototype neuronal promoter as it is moderately expressed and has a well characterised restricted expression pattern in the CNS. Characterisation of the 5'- romoter region of the GABAA receptor 6 subunit gene was achieved by sequencing 10.5 kb of DNA from the 5'-upstream region. Analysis of this sequence revealed the presence of several putative recognition sites for transcription factors. Identification of the transcription start points showed two main clusters of start sites located 58 and 108 bases upstream from the translational start point. Comparison of about 3.5 kb of DNA sequence of the promoters of the rat and mouse genes showed a high degree of conservation of noncoding sequence between the species. The recognition site for a putative regulatory factor BSF 1 identified in the 5'-upstream sequence of the rat delta gene was demonstrated to be absent in the mouse 5'-flanking sequence. Analysis of a range of promoter - reporter constructs in the NB4 1A3 cell line suggested the presence of a silencing element located between 4.5 kb and 6.3 kb upstream of the translational start point. As a preliminary step to evaluating in vivo mutagenesis investigations of GABAA receptor 5 subunit gene promoter function, targeted disruption of the GABAA receptor 6 subunit gene in embryonic stem cells was achieved. Two genomic DNA fragments from the GABAA receptor 5 subunit gene were selected and inserted into a simple replacement vector (pNT) containing the neomycin gene and the HSV thymidine kinase gene. A successful targeting event would result in the removal of the first exon, 4.5 kb of promoter sequence and 2 kb of DNA from the first intron. Two targeted embryonic stem cell lines were isolated and can be used to make mice homozygous for the mutated gene. The GABAA receptor delta subunit gene had previously been assigned to human Chromosome Ip. Genetic mapping of the gene to rat chromosome 5 and mouse chromosome 4 was performed. These results agree with expected regions of synteny between human, mouse and rat. A range of recombinant HSV-1 viruses were produced which contained the E coli lacZ gene driven by different promoters. Two loci within the HSV-1 genome were chosen as sites of insertion, LAT and UL43 in two HSV-1 viral variants, 1716 and 1764. Infection of primary cerebellar granule cell cultures with a range of these recombinant viruses revealed that the cerebellar granule cells were not readily infected by this viral variant. Stereotactic injection of the viruses into the cerebellum of adult rats failed to show specific P-galactosidase expression. Nevertheless, the GABAA receptor delta subunit gene promoter-lacZ fusion constructs can now be transferred to new HSV-1 vectors with better growth characteristics thus furthering the original goal of this work.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available