Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/17363
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Type: Journal article
Title: Over-expression of human lysosomal -mannosidae in mouse embryonic stem cells
Author: Robinson, A.
Crawley, A.
Hopwood, J.
Citation: Molecular Genetics and Metabolism, 2005; 85(3):203-212
Publisher: Academic Press Inc Elsevier Science
Issue Date: 2005
ISSN: 1096-7192
1096-7206
Abstract: Alpha-mannosidosis is a lysosomal storage disorder characterised by the lysosomal accumulation of mannose-containing oligosaccharides and a range of pathological consequences, caused by a deficiency of the lysosomal enzyme alpha-mannosidase. One of the major features of alpha-mannosidosis is progressive neurological decline, for which there is no safe and effective treatment. Implantation of stem cells into the central nervous system has been proposed as a potential therapy for these disorders. We report the construction and characterisation of mouse embryonic stem cell lines for the sustained over-expression of recombinant human lysosomal alpha-mannosidase (rhalphaM). Two vectors (involving recombinant human alpha-mannosidase expression driven by either the chicken beta-actin promoter/CMV enhancer or by the elongation factor 1-alpha promoter) were constructed and used to transfect mouse D3 embryonic stem cells. Selected clonal cell lines were isolated and tested to evaluate their expression of recombinant human alpha-mannosidase. Stem cell clones transfected with the chicken beta-actin promoter/CMV enhancer maintained rhalphaM expression levels throughout differentiation. This expression was not markedly elevated above background. In contrast, the vector incorporating the elongation factor 1-alpha promoter facilitated substantial over-expression of alpha-mannosidase when analysed out to 21 days of differentiation in stably transfected cell lines. The highest expressing cell line was found to qualitatively retain a similar differentiation potential to untransfected cells, and to secrete alpha-mannosidase that could mediate a reduction in the level of oligosaccharides stored by human alpha-mannosidosis skin fibroblasts. These results suggest potential for the use of this cell line for investigation of a stem cell therapy approach to treat alpha-mannosidosis.
Keywords: Cell Line; Stem Cells; Animals; Humans; Mice; alpha-Mannosidosis; alpha-Mannosidase; Mannosephosphates; Stem Cell Transplantation; Transfection; Gene Expression; Up-Regulation
RMID: 0020050686
DOI: 10.1016/j.ymgme.2005.03.005
Appears in Collections:Paediatrics publications

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