Tseng W C, Haselton F R, Giorgio T D
Department of Chemical, Vanderbilt University, Nashville, Tennessee 37235, USA.
J Biol Chem. 1997 Oct 10;272(41):25641-7. doi: 10.1074/jbc.272.41.25641.
Cationic liposomes are potentially important gene transfer vehicles, although their application has been limited by relatively low efficiency of transgene expression. Single cell quantitative methods, such as those used in this study, should permit a more detailed understanding of the relationships between delivered plasmid and transgene expression. Intracellular plasmid delivery and transgene expression were measured simultaneously using photoconjugated ethidium monoazide as an intracellular plasmid delivery marker and green fluorescent protein (GFP(S65T)) as a transgene expression marker. Quantitative flow cytometry was used to estimate plasmid copy number and GFP(S65T) molecules in single cells. The plasmid was delivered to HeLa cells with a cationic liposome vehicle containing 1,2-dioleoyloxy-3-trimethylammonium-propane and dioleoylphosphatidylethanolamine (1:1 mol/mol). Treatment was carried out continuously for 24 h. Flow cytometry measurements on 20, 000 cells were performed during treatment and for 48 h post-treatment. On a single cell basis, transgene expression efficiency and average GFP(S65T) expression level increased with intracellular plasmid copy number. After 3-h exposure to the liposomal vector, more than 95% of the cells were positive for plasmid entry, but none had detectable transgene expression. Maximum transgene expression was achieved at 24 h and remained unchanged at the 72-h measurement. At 24 h, the average positive cell contained 1.6 x 10(5) plasmid copies and 2.3 x 10(6) GFP(S65T) molecules. Importantly, the measurement strategies revealed that transgene expression varied widely within the entire cell population. Although only 30% of all cells expressed transgene, the subpopulation of cells that rapidly incorporated the vector demonstrated 100% efficiency in transgene expression. This study identifies parameters that modulate highly efficient transgene expression from plasmid delivery by cationic liposomes.
阳离子脂质体是潜在的重要基因传递载体，尽管其应用受到转基因表达效率相对较低的限制。单细胞定量方法，如本研究中使用的方法，应能更详细地了解导入质粒与转基因表达之间的关系。使用光共轭单叠氮溴化乙锭作为细胞内质粒传递标记物，绿色荧光蛋白（GFP(S65T)）作为转基因表达标记物，同时测量细胞内质粒传递和转基因表达。采用定量流式细胞术估计单细胞中的质粒拷贝数和GFP(S65T)分子数。将质粒与含有1,2 - 二油酰氧基 - 3 - 三甲基铵丙烷和二油酰磷脂酰乙醇胺（1:1摩尔/摩尔）的阳离子脂质体载体一起递送至HeLa细胞。连续处理24小时。在处理期间和处理后48小时对20000个细胞进行流式细胞术测量。在单细胞水平上，转基因表达效率和平均GFP(S65T)表达水平随细胞内质粒拷贝数增加而增加。暴露于脂质体载体3小时后，超过95%的细胞质粒进入呈阳性，但无一细胞有可检测到的转基因表达。在24小时时达到最大转基因表达，并且在72小时测量时保持不变。在24小时时，平均阳性细胞含有1.6×10⁵个质粒拷贝和2.3×10⁶个GFP(S65T)分子。重要的是，测量策略表明整个细胞群体中转基因表达差异很大。虽然所有细胞中只有30%表达转基因，但快速摄取载体的细胞亚群转基因表达效率达100%。本研究确定了调节阳离子脂质体介导的质粒传递产生高效转基因表达的参数。
