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Heterologous expression of wildtype and mutant myocilin in High Five™ insect cells shows comparable effects to cultivated trabecular meshwork cells

Paper ID Volume ID Publish Year Pages File Format Full-Text
14153 1177 2007 5 PDF Available
Title
Heterologous expression of wildtype and mutant myocilin in High Five™ insect cells shows comparable effects to cultivated trabecular meshwork cells
Abstract

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™). 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.

Keywords
Myocilin; MYOC; TIGR
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Heterologous expression of wildtype and mutant myocilin in High Five™ insect cells shows comparable effects to cultivated trabecular meshwork cells
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biomolecular Engineering - Volume 24, Issue 3, September 2007, Pages 313–317
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us