Phosphate-enhanced transfection of cationic lipid-complexed mRNA and plasmid DNA
Phosphate-enhanced transfection of cationic lipid-complexed mRNA and plasmid DNA
复制标题
DOI：
10.1016/s0005-2736(97)00238-1
复制
发表时间：
1998-03-02
影响因子：
3.4
通讯作者：
Langer, DJ
中科院分区：
文献类型：
--
作者：
Karikó, K;Kuo, A;Langer, DJ
阳离子脂质介导的基因转移已被证明是一种有效的促进细胞基因转移的机制。提高阳离子脂质介导的转基因表达效能的一种方法是优化脂质与核酸之间形成复合物的条件。在本报告中，我们描述了在与质粒DNA或mRNA络合之前，使用磷酸盐缓冲液预处理脂溶素（N-[1-（2,3-二聚氧基）丙基]- N， N， N-三甲基氯化铵（DOTMA）和二油基磷脂酰乙醇胺（DOPE）的1:1 （w/w）混合物）的有益效果。在这种优化条件下，我们研究了DNA和rna介导的转基因在人骨肉瘤细胞系（HOS）中的表达动力学，脂素在磷酸盐缓冲液中预孵育导致质粒DNA和mRNA中荧光素酶的表达分别增加了26倍和56倍。添加氯喹（50 μ M）可将质粒介导的基因传递增强3倍，并与磷酸盐协同作用，使荧光素酶的表达增加46倍。磷酸盐预孵育既缩短了细胞摄取所需的时间，也缩短了实现最大转基因表达的时间。最佳转染条件为30-50 mM磷酸，pH 5.6-6.8，磷酸阴离子为二价。磷酸阴离子的作用是特异性的，单价的氯离子和醋酸离子没有刺激作用。这些结果表明，当阳离子脂质与带负电荷的核酸和细胞膜接触时，二价磷酸阴离子在复合物形成和转染过程中起刺激作用。这些发现描述了显著提高DNA和mRNA转染效率的特定条件，并为基因转染研究提供了有效的程序。(C) 1998爱思唯尔科学有限公司
Cationic lipid-mediated gene transfer has been shown to be a competent albeit inefficient mechanism of promoting cellular gene transfer. One way to improve the efficacy of cationic lipid-mediated transgene expression is to optimize conditions for complex formation between the lipids and nucleic acids. In this report we describe the beneficial effects of using phosphate buffer to precondition lipofectin (a 1:1 (w/w) mixture of N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA), and dioleoyl phosphatidylethanolamine (DOPE)) prior to complexing with plasmid DNA or mRNA. Under such optimized conditions we studied the kinetics of DNA- and RNA-mediated transgene expression in a human osteosarcoma cell line (HOS), Preincubation of lipofectin in phosphate buffer resulted in up to 26- and 56-fold increases in luciferase expression from plasmid DNA and mRNA, respectively. Addition of chloroquine (50 mu M), which enhanced plasmid-mediated gene delivery 3-fold, was synergistic with phosphate resulting in an additional 46-fold increase in luciferase expression. The preincubation with phosphate shortened both the time required for cellular uptake and the time to achieve maximal transgene expression. Optimal transfection was achieved in the presence of 30-50 mM phosphate, at pH 5.6-6.8 under which the phosphate anion is divalent. The effect of phosphate anion was specific in that monovalent Cl- and acetate anions were not stimulatory. These results demonstrate that divalent phosphate anion plays a stimulatory role during complex formation and transfection when cationic lipids come in contact with negatively charged nucleic acids and cell membranes. These findings delineate specific conditions which dramatically enhance transfection efficiency for both DNA and mRNA, and provide an effective procedure for gene transfection studies. (C) 1998 Elsevier Science B.V.