|Title:||Characterisation of a gene critical in eye and lens development|
|Publisher:||University of Sydney.|
Children's Medical Research Institute.
Children's Hospital at Westmead Clinical School.
|Abstract:||Visual impairment or blindness caused by vision disorders affect over 500,000 Australians, with approximately one-third caused by genetic eye disorders. Previous work identified a cataract and anterior segment abnormality patient with a de novo balanced chromosomal translocation transecting the 5’ UTR of a novel candidate disease gene. An expression vector containing the candidate gene and a GFP tag revealed its subcellular localisation to the plasma membrane domain of two epithelial cell lines. Colocalisation experiments showed colocalisation of the candidate disease gene with proteins of adherens junctions, tight junctions and cytoskeletal F-actin in Caco2 cells. Caco2 cells transfected with a mutation in the candidate disease gene caused clustering of F-actin stress fibres in the basal regions of the cell, suggesting the presence of functional actin-binding domains in this candidate disease gene. Stable knockdown cells when cultured in a 3D-cell assay produced abnormal cyst structures characterised by the presence of additional ectopic lumens, multilayering of cells and misexpression of a polarity marker. Knockdown cells generated a significantly greater number of abnormal cysts. The phenotype exhibited by knockdown cell lines is suggestive of defects affecting the actin cytoskeleton and polarity. To further investigate the role of the candidate disease gene in eye development, gene trap loss-of-function mice were generated and examined. Homozygous loss-of-function mice developed congenital cataracts and demonstrated structural lens abnormalities and abnormalities in the expression of cell adhesion and polarity proteins in the developing lens. The lens abnormalities exhibited by the mutant mice, along with the defects observed in the mutant and knockdown cell assays suggests that this novel candidate disease gene has an important role in the regulation of the actin cytoskeleton and cellular polarity in epithelial cells and lens development.|
|Access Level:||Access is restricted to staff and students of the University of Sydney . UniKey credentials are required. Non university access may be obtained by visiting the University of Sydney Library.|
|Rights and Permissions:||The author retains copyright of this thesis. It may only be used for the purposes of research and study. It must not be used for any other purposes and may not be transmitted or shared with others without prior permission.|
|Type of Work:||PhD Doctorate|
|Type of Publication:||Doctor of Philosophy Ph.D.|
|Appears in Collections:||Sydney Digital Theses (University of Sydney Access only)|
|GREENLEES Rebecca - Final thesis.pdf||22.87 MB||Adobe PDF|
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