Efficiency of Membrane Protein Expression Following Infection with Recombinant Adenovirus of Polarized Non-Transformed Human Retinal Pigment Epithelial Cells.
Efficiency of Membrane Protein Expression Following Infection with Recombinant Adenovirus of Polarized Non-Transformed Human Retinal Pigment Epithelial Cells.
复制标题
极化非转化人视网膜色素上皮细胞重组腺病毒感染后膜蛋白表达的效率。
DOI：
10.1007/978-3-319-17121-0_97
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发表时间：
2016
影响因子：
--
通讯作者：
Finnemann,SilviaC
中科院分区：
文献类型：
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作者：
Müller,Claudia;Blenkinsop,TimothyA;Stern,JeffreyH;Finnemann,SilviaC
外源性蛋白质的瞬时表达有助于研究培养的视网膜色素上皮（RPE）细胞移植的分子机制和实用性。在此，我们比较了在两种最近建立的分化的人RPE模型，成人RPE干细胞衍生的RPE和原代胎儿RPE中，在用重组腺病毒感染或用脂质体中的DNA转染后膜蛋白β5整合素-GFP（β5-GFP）的表达。我们改变病毒滴度和病毒孵育时间，并通过共聚焦显微镜检查操作细胞中的β5-GFP和紧密连接标记ZO-1。在脂质体介导的转染后，少于5%的细胞表达β5-GFP。在腺病毒感染短至1 h后，可检测到β5-GFP的细胞百分比超过90%。将病毒滴度降低两倍并不改变表达β5-GFP的细胞的分数，但增加了细胞间β5-GFP水平的变异性。在低表达水平的细胞中，β5-GFP主要定位于顶端质膜，如内源性αvβ5整合素。在具有高表达水平的细胞中，β5-GFP定位于细胞质以及顶端表面，表明在运输隔室中积累。总之，腺病毒递送在培养的分化的人RPE细胞中产生正确极性的有效外源性膜蛋白表达。
Transient expression of exogenous proteins facilitates studies of molecular mechanisms and utility for transplantation of retinal pigment epithelial (RPE) cells in culture. Here, we compared expression of the membrane protein β5 integrin-GFP (β5-GFP) in two recently established models of differentiated human RPE, adult RPE stem cell-derived RPE and primary fetal RPE, upon infection with recombinant adenovirus or transfection with DNA in liposomes. We varied viral titer and duration of virus incubation and examined β5-GFP and the tight junction marker ZO-1 in manipulated cells by confocal microscopy. Fewer than 5 % of cells expressed β5-GFP after liposome-mediated transfection. The percentage of cells with detectable β5-GFP exceeded 90 % after adenovirus infection for as little as 1 h. Decreasing virus titer two-fold did not alter the fraction of cells expressing β5-GFP but increased variability of β5-GFP level among cells. In cells with low expression levels, β5-GFP localized mostly to the apical plasma membrane like endogenous αvβ5 integrin. In cells with high expression levels, β5-GFP localized to the cytoplasm in addition to the apical surface suggesting accumulation in trafficking compartments. Altogether, adenovirus delivery yields efficient exogenous membrane protein expression of correct polarity in differentiated human RPE cells in culture.