The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids
The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids
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DOI：
10.1016/s0014-5793(97)00973-3
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发表时间：
1997-09-08
期刊：
影响因子：
3.5
通讯作者：
Vandenbranden, M
中科院分区：
文献类型：
--
作者：
ElOuahabi, A;Thiry, M;Vandenbranden, M
我们使用P-32标记的pCMV-CAT质粒DNA来估计DNA摄取效率，并使用未标记的pCMV-CAT质粒DNA来定量使用以下三种阳离子化合物中的每一种转染COS细胞后的CAT活性：（3-十四烷基氨基-N-叔丁基-N '-十四烷基丙脒，和先前描述的二C14-脒[1]，[2]脂质体转染素（a 1：1 N-（1- 2，3-二油基氧基丙基）-N，N，N-三乙基铵（DOTMA）和二油基磷脂-乙醇胺（DOPE）的混合物），和[3] DMRIE-C（答1：1 N-[1-（2，3-二肉豆蔻基氧基）丙基]-N，N-二甲基-N-（2-羟基-乙基）溴化铵（DMRIE）和胆固醇）。令人惊讶的是，用维他米定观察到高CAT活性，尽管与脂质体和DMRIE-C相比DNA摄取效率较低，透射电子显微镜（TEM）显示，内吞作用是这三种阳离子脂质进入COS细胞的主要途径，但与含有vectamidine或DMRIE-C的复合物接触的内体膜常表现出破坏的形态。用DNA-脂质转染素复合物观察到的这种内体破坏要少得多。三种化合物的这种比较表明，由阳离子脂质介导的有效转染不仅与它们的摄取百分比相关，而且与它们使内体不稳定和从内体逃逸的能力相关。(C)1997年欧洲生物化学学会联合会。
We used a P-32-labeled pCMV-CAT plasmid DNA to estimate the DNA uptake efficiency and unlabeled pCMV-CAT plasmid DNA to quantify the CAT activity after transfection of COS cells using each of the three following cationic compounds: [1] vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecyl propionamidine, and previously described as diC14-amidine [1], [2] lipofectin (a 1:1 mixture of N-(1-2,3-dioleyloxypropyl)-N,N,N-triethylammonium (DOTMA) and dioleylphosphatid-ylethanolamine (DOPE)), and [3] DMRIE-C (a 1:1 mixture of N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxy-ethyl) ammonium bromide (DMRIE) and cholesterol), Surprisingly, a high CAT activity was observed with vectamidine although the DNA uptake efficiency was lower as compared to lipofectin and DMRIE-C, Transmission electron microscopy (TEM) revealed endocytosis as the major pathway of DNA-cationic lipid complex entry into COS cells for the three cationic lipids, However, the endosomal membrane in contact with complexes containing vectamidine or DMRIE-C often exhibited a disrupted morphology. This disruption of endosomes was much less frequently observed with the DNA-lipofectin complexes, This comparison of the three compounds demonstrate that efficient transfection mediated by cationic lipids is not only correlated to their percentage of uptake but also to their ability to destabilize and escape from endosomes. (C) 1997 Federation of European Biochemical Societies.