Gaucher disease, an autosomal recessive disorder, is characterized by a heterogeneous set of signs and symptoms caused by a deficiency in the lysosomal enzyme glucocerebrosidase. As a single gene enzyme deficiency, Gaucher disease is a prime candidate for enzyme replacement therapy. Such therapy exists, though the exorbitant cost prevents many fiom receivii treatment. Thus, a more cost-effective method of producing glucocerebrosidase was examined. The Pichiapastoris yeast system was chosen, but resultant production levels were low. Two variants of green fluorescent protein (GFP), red-shifted GFP (RSGFP) and enhanced GFP (EGFP), were employed as molecular reporters to track enzyme production and isolation. No expression of glucocerebrosidase was evident, indicating that the P. pastoris system was not an appropriate choice for glucocerebrosidase production. Both GFP variants were successfully expressed, with EGFP levels apparently greater than RSGFP levels. To study glucocerebrosidase production and trafficking in a higher eukaryotic system, EGFP-tagged glucocerebrosidase constructs were expressed in HeLa cells. Though EGFP was readily visualized, few cells expressing glucocerebrosidase constructs were present. No co-localization with organelle markers was evident. Examination at the RNA level indicated successfid transcription, however, an apparent translational inefficiency was encountered. To shed light on the possible cause of this inefficiency, two approaches were taken: one examined expression of truncated glucocerebrosidase constructs in HeLa cells, the other included co-transfection with small interfering RNAs (siRNAs) in both HeLa and COS- 1 cells. In the first approach, greater expression was seen itom the EGFPtagged construct devoid of the proposed inhibitory binding site than itom the EGFPtagged construct containing the binding site. Expression of both truncated constructs was greater than that of EGFP-tagged glucocerebrosidase starting at either initiation codon, indicating a more complex mechanism of translational control than strictly inhibition fiom the proposed site. In the second approach, a siRNA was designed to block TCP80, which has been suggested to inhibit glucocerebrosidase translation. Co-transfection studies of siJ3NAs (control, EGFP and TCP80) and glucocerebrosidasel EGFP plasmids were performed in HeLa and COS-1 cells. In both cell types, all constructs were successfblly expressed when co-transfected with control siRNA, as indicated by RNA and protein examination. Introduction of TCP80 siRNA in both cell types did not serve to increase glucocerebrosidase expression as expected, but instead decreased such expression. EGFP expression was readily knocked down in HeLa and COS-1 cells by GF'P-targeted siRNk Knockdown was evident in the expression of glucocerebrosidase/EGFP constructs, indicating that hsion with EGFP may serve as a means to introduce a foreign gene, then knock its expression down at a desired time by introduction of a GFP-targeted siRNA.
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/354 |
| Date | 10 April 2008 |
| Creators | Campbell, Tessa Nicole. |
| Contributors | Choy, Francis Y. M. |
| Source Sets | University of Victoria |
| Detected Language | English |
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