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Title: Use of adeno-associated virus (AAV) to investigate transcriptional regulation of the preprotachykinin-A (PPT-A) promoter in cultured DRG neurons
Author: Gerrard, L.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2002
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This study set out to investigate transcriptional regulation of the rat preprotachykinin-A (PPT-A) gene promoter in dorsal root ganglia neurons (DRG). The PPT-gene is regulated by a variety of extracellular stimuli including NGF (Lindsay and Harmer, 1989), GDNF (Ogun-Muyiwa et al., 1999), steroids (Smith et al., 1991), and inflammation (Noguchi and Ruda, 1992) and it is likely that many of these stimuli act at the level of transcription. The proximal promoter region of the PPT-A gene has been extensively studied (Fiskerstrand and Quinn, 1996) however functional studies have been greatly hindered due to the lack of clonal cell lines which express endogenous PPT-A or can support PPT-A promoter function in reporter gene constructs. DRG neurons are generally refractory to all methods of transfection therefore adeno-associated virus vectors (AAV) were used in a tool for the transduction of PPT-A promoter fragments driving a receptor gene to investigate PPT-A promoter activity. The first step involved the optimisation of AAV vector production and infection of heterogenous cultures of DRG. This was achieved using AAV vectors and contained either the CMV promoter or the PPT-A proximal promoter (spanning nucleotides -865 to +92) driving the GFP reporter gene. GFP expression was visualised and it was established that when high titres of AAV vectors were applied, those containing the CMV promoter resulted in 100% transduction efficiency of both neuronal and nonneuronal cells and those containing the PPT-A promoter demonstrated a more restricted expression pattern. In addition, both promoter fragments allowed long-term reporter gene expression in cultured DRG neurons in which expression was predominately neuronal.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available