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首页> 外文期刊>Biomolecular engineering >Heterologous expression of wildtype and mutant myocilin in High Five (TM) insect cells shows comparable effects to cultivated trabecular meshwork cells
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Heterologous expression of wildtype and mutant myocilin in High Five (TM) insect cells shows comparable effects to cultivated trabecular meshwork cells

机译：在High Five（TM）昆虫细胞中野生型和突变型myocilin的异源表达显示出与培养的小梁网状细胞相当的效果

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摘要

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 (TM)). 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. (C) 2007 Elsevier B.V. All rights reserved.
机译：Myocilin（MYOC，TIGR）变异与青少年和原发性开角型青光眼（POAG）相关。为了调查MYOC野生型过表达和所选突变的后果，我们建立了异源昆虫细胞系统（High Five（TM））。将野生型，Pro370Leu，Gln368X和Lys423Glu克隆到修饰的pIB / V5-His（pEXIV）载体中，在框架融合中有无下游GFP。通过体外诱变引入突变。显示了异源表达并通过RT-PCR，蛋白质印迹，免疫细胞化学和荧光显微镜分析。延长的培养时间（> 14天）导致所有粗粒内质网扩张水箱中所有变体的MYOC蛋白积累。最后，发生过表达的野生型和突变体的细胞死亡。通过电子显微镜观察到核糖体在细胞死亡之前直接附着在这些生长的囊泡上。我们的观察结果表明，该系统适用于追踪MYOC突变体的细胞内作用。 （C）2007 Elsevier B.V.保留所有权利。

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