Marden, Chloe Maria; (1999) Functional analysis of the p47phox gene promoter. Doctoral thesis (Ph.D), UCL (University College London).

Text out.pdf Download (20MB)

Abstract

Defects in the p47phox gene that encodes a cytosolic component of the phagocytic NADPH oxidase complex are responsible for the majority of cases of autosomal recessive Chronic Granulomatous Disorder (AR-CGD). Somatic gene therapy for p47phox-deficient AR-CGD requires regulated, sustainable expression of in cells affected by CGD. This thesis investigated regulation of the gene with the ultimate aim of constructing a retroviral vector that incorporates p47phox regulatory sequence. Myeloid and non-myeloid cell lines were transfected with CAT reporter gene constructs containing 57-3100 base pairs (bp) of the p47phox 5' promoter. Assay of CAT activity in stable transfectants demonstrated that -3100bp promoter was insufficient to direct fully regulated expression. However, an inhibitory region located upstream of position -500, and proximal sequence (-143bp) sufficient for the direction of significant expression in myeloid cells were identified. Mutation analysis demonstrated that an interferon-stimulated response element (ISRE) located between positions -103 and -116 and a consensus binding site for the Ets family transcription factor PU.1 located between positions -38 and -43 were required for optimal expression in myeloid cells. Electrophoretic mobility shift assay (EMSA) demonstrated myeloid and B-cell-specific binding of PU.1 at the consensus binding site. EMSA revealed that PU.1 bound as four distinct species in a cell-type-specific pattern that altered significantly in response to myeloid differentiation. Phosphatase treatment of nuclear extract suggested that PU.1 EMSA complexes were differentially phosphorylated. Based on the results of functional and protein binding studies, a -143bp p47phox promoter fragment containing ISRE and PU.1 motifs was incorporated into a U3-deleted retroviral vector and stably transfected into a retroviral packaging line to produce a replication-deficient p47phox CAT reporter retrovirus. Transduction of myeloid and non-myeloid cell lines resulted in significant CAT reporter gene activity in myeloid cells.

Download activity - last month

Export as XMLCSVJSON

Download activity - last 12 months

Export as XMLCSVJSON

Downloads by country - last 12 months

Export as CSVXMLJSON

Archive Staff Only

View Item