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Abstract #19807 Published in IGR 9-4
Heterologous expression of wildtype and mutant myocilin in High Five™ insect cells shows comparable effects to cultivated trabecular meshwork cells
Oezbey S; Stengel C; Schlotzer-Schrehardt U; Ekici A; Rautenstrauss BBiomolecular Engineering 2007; 24: 313-317
Myocilin (MYOC, TIGR) variations are associated with juvenile and primary open-angle glaucoma (POAG). To investigate consequences of MYOC wildtype overexpression and selected mutations, we established a heterologous insect cell system (High Five(trademark)). Wildtype, Pro370Leu, Gln368X and Lys423Glu were cloned into a modified pIB/V5-His (pEXIV) vector with and without downstream GFP in frame fusion. Mutations were introduced by in vitro mutagenesis. Heterologous expression was shown and analysed by RT-PCR, Western blotting, immunocytochemistry and fluorescence microscopy. Extended cultivation (> 14 days) resulted in accumulation of MYOC protein for all variants in growing dilated cisterns of the rough endoplasmic reticulum. Finally cell death for overexpressed wildtype and mutants occurs. A direct attachment of ribosomes to these growing vesicles preceding the cell death was observed by electron microscopy. Our observations indicate that this system is suitable to trace the intracellular effects of MYOC mutants.
Dr. B. Rautenstrauss, University Hospital Erlangen, Institute of Human Genetics, Schwabachanlage 10, 91054 Erlangen, Germany. Bernd.Rautenstrauss@humgenet.uni-erlangen.de
Classification:
3.6 Cellular biology (Part of: 3 Laboratory methods)
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
2.5.1 Trabecular meshwork (Part of: 2 Anatomical structures in glaucoma > 2.5 Meshwork)