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Title: The development of improved GFP mutants : protein tagging and visualisation of gene expression patterns in mammalian cells and transgenic mice
Author: Her, Guor Mour
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
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To increase fluorescent activity of GFP in the mammalian environment, mutations were created in a humanised form of GFP (containing mammalian codon-usage preferences). The fluorescence and expression efficiency of these mutants was characterised in mammalian cells and three of them showed strong fluorescence. In addition, stable green fluorescence was seen in established cell lines expressing these mutants with no obvious detrimental effect. In a study of WT1 nuclear function, three distinct nuclear patterns were seen in Cos7 cells depending on the isoform of WT1 in the GFP-WT1 fusions. These patterns were very similar to those reported for native forms of WT1 and the fluorescent signal provided a means to study these patterns at a high resolution. Functional domain analysis of WT1 was performed by expressing GFP and truncated WT1 fusions which suggested that the C-terminus (zinc finger regions) of WT1 had nuclear localisation signal(s) and additional signals which determine the specificity of the nuclear pattern. Altered nuclear patterns of GFP-WT1 fusion implied that there was an association between native and truncated WT1 which may result in a dominant negative behaviour and the mutated proteins caused dysfunctions of WT1 in cells. GFP transgenic mice were generated containing double-reporter constructs with either GFP fused to β-galactosidase or a dicistronic gene expressing GFP and β-galactosidase by a viral IRES sequence. Expressions of these were driven by the Msx-1 promoter. Five GFP transgenic lines were generated and expressed both GFP fluorescence and x-gal enzymatic activity in embryos from E9.5 to E12.5. Both reporter activities were seen in many embryonic tissues including maxillary and mandibular arches, eye, dorsal neural tube, somites, limbs and mid brain.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available