Use of a quaternary ammonium detergent in liposome mediated DNA transfection of mouse L-cells.
Use of a quaternary ammonium detergent in liposome mediated DNA transfection of mouse L-cells.
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在脂质体介导的小鼠 L 细胞 DNA 转染中使用季铵去污剂。
DOI：
10.1016/0005-2736(89)90099-0
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发表时间：
1989
期刊：
影响因子：
--
通讯作者：
Huang,L
中科院分区：
文献类型：
--
作者：
Pinnaduwage,P;Schmitt,L;Huang,L
由二油酰磷脂酰乙醇胺 (DOPE) 和季铵去污剂（十二烷基、十四烷基或十六烷基三甲基溴化铵）组成的超声处理脂质体介导 pSV2 CAT 质粒 DNA 向小鼠 L929 成纤维细胞的功能性转移。通过测定接触脂质-DNA 复合物 40 小时后收集的细胞裂解物中的氯霉素乙酰转移酶活性来确定转染是否成功。用季铵去污剂制备的脂质体比相同浓度的游离去污剂毒性更小，并且在将质粒DNA递送至细胞方面更有效。对三种去垢剂与脂质的组合分析表明，十六烷基三甲基溴化铵对细胞的毒性最小。这种去垢剂在 DOPE 中的最低浓度为 20 mol%，可实现稳定的脂质体制剂和有效的转染。使用 30 μg DNA 可获得最佳转染效率。 DNA 浓度的进一步增加导致转染效率降低，这可能是由于现在充满带负电荷的 DNA 的脂质体与带负电荷的细胞表面之间的电荷排斥所致。脂质体的转染活性受到其在高脂质体浓度下的细胞毒性的限制。将这些结果与 Lipofectin（另一种市售的带正电荷的脂质体制剂）的结果进行比较。尽管含有季铵去污剂的脂质体的总体转染活性略低于Lipofectin，但它可以作为一种廉价且方便的替代品。
Sonicated liposomes composed of dioleoylphosphatidylethanolamine (DOPE) and a quaternary ammonium detergent (dodecyl-, tetradecyl-, or cetyl-trimethylammonium bromide) mediates functional transfer of pSV2 CAT plasmid DNA to mouse L929 fibroblasts. Successful transfection was determined by assaying for chloramphenicol acetyltransferase activity in cell lysates collected 40 h after exposure to the lipid-DNA complexes. Liposomes prepared with the quaternary ammonium detergents were less toxic than the free detergents at the same concentrations and were more efficient in their delivery of the plasmid DNA to the cells. Analysis of the three detergents in combination with the lipid showed that cetyltrimethylammonium bromide was least toxic to the cells. This detergent, at a minimal concentration of 20 mol% in DOPE, allowed for stable liposome preparations and efficient transfection. Optimal efficiency of transfection occurred with 30 μg of DNA. Further increases in the DNA concentration caused a decrease in the transfection efficiency, perhaps due to charge repulsions between the liposomes now saturated with negatively charged DNA and the negatively charged cell surface. The transfection activity of the liposome was limited by its cytotoxicity at high liposome concentrations. These results are compared with that of the Lipofectin, another positively charged liposome preparation which is commercially available. Although the overall transfection activity of the liposome containing the quaternary ammonium detergent is somewhat lower than that of the Lipofectin, it may serve as an inexpensive and convenient alternative.