Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipids.
Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipids.
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使用有效脂质瞬时转染带有 CFTR 和报告基因的极化上皮单层。
DOI：
10.1152/ajpcell.00435.2002
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Schwiebert,LisaM
中科院分区：
文献类型：
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作者：
Tucker,TorryA;Varga,Karoly;Bebok,Zsuzsa;Zsembery,Akos;McCarty,NaelA;Collawn,JamesF;Schwiebert,ErikM;Schwiebert,LisaM
用脂质试剂瞬时转染上皮细胞由于毒性和缺乏功效而受到限制。在这项研究中，我们表明，最近开发的脂质体抑制非极化的人气道上皮细胞具有高功效和效率，几乎没有或没有毒性。由于这一成功，我们假设这些脂质也可能允许瞬时转染极化上皮单层。检测了一组试剂将报告基因荧光素酶（LUC）转移到非囊性纤维化（非CF）和CF人支气管上皮细胞、MDCK上皮细胞单层以及最终原代非CF和CF气道上皮细胞的极化单层中。脂质试剂，这是最成功的initialLUCassays，也进行了测试，为载体的报告基因携带绿色荧光蛋白（GFP）的转移和成功的转染和表达的上皮特异性蛋白，囊性纤维化跨膜传导调节（CFTR）。进行电生理学、生物化学和免疫学测定以显示瞬时表达CFTR的上皮单层的成功互补。我们还提出了有助于促进这些气道上皮细胞系单层形成的研究结果。总之，这些数据表明，极化单层细胞被最近开发的脂质（特别是LipofectAMINE PLUS和LipofectAMINE 2000）瞬时转染。上皮细胞单层的瞬时转染提供了一个强大的系统，在该系统中，在天然极化上皮细胞中瞬时表达任何上皮特异性蛋白的cDNA，以研究蛋白质功能。
Transient transfection of epithelial cells with lipid reagents has been limited because of toxicity and lack of efficacy. In this study, we show that more recently developed lipids transfect nonpolarized human airway epithelial cells with high efficacy and efficiency and little or no toxicity. Because of this success, we hypothesized that these lipids may also allow transient transfection of polarized epithelial monolayers. A panel of reagents was tested for transfer of the reporter gene luciferase (LUC) into polarized monolayers of non-cystic fibrosis (non-CF) and CF human bronchial epithelial cells, MDCK epithelial cell monolayers, and, ultimately, primary non-CF and CF airway epithelial cells. Lipid reagents, which were most successful in initialLUCassays, were also tested for transfer of vectors bearing the reporter gene green fluorescent protein (GFP) and for successful transfection and expression of an epithelial-specific protein, the cystic fibrosis transmembrane conductance regulator (CFTR). Electrophysiological, biochemical, and immunological assays were performed to show successful complementation of an epithelial monolayer with transiently expressed CFTR. We also present findings that help facilitate monolayer formation by these airway epithelial cell lines. Together, these data show that polarized monolayers are transfected transiently with more recently developed lipids, specifically LipofectAMINE PLUS and LipofectAMINE 2000. Transient transfection of epithelial monolayers provides a powerful system in which to express the cDNA of any epithelium-specific protein transiently in a native polarized epithelium to study protein function.