Baker A, Cotten M
Institute for Molecular Pathology (IMP), Dr BohrGasse 7, 1030 Vienna, Austria.
Nucleic Acids Res. 1997 May 15;25(10):1950-6. doi: 10.1093/nar/25.10.1950.
Molecular biology has many applications where the introduction of large (>100 kb) DNA molecules is required. The current methods of large DNA transfection are very inefficient. We reasoned that two limits to improving transfection methods with these large DNA molecules were the difficulty of preparing workable quantities of clean DNA and the lack of rapid assays to determine transfection success. We have used bacterial artificial chromosomes (BACs) based on the Escherichia coli F factor plasmid system, which are simple to manipulate and purify in microgram quantities. Because BAC plasmids are kept at one to two copies per cell, the problems of rearrangement observed with YACs are eliminated. We have generated two series of BAC vectors bearing marker genes for luciferase and green fluorescent protein (GFP). Using these reagents, we have developed methods of delivering BACs of up to 170 kb into mammalian cells with transfection efficiency comparable to 5-10 kb DNA. Psoralen-inactivated adenovirus is used as the carrier, thus eliminating the problems associated with viral gene expression. The delivered DNA is linked to the carrier virus with a condensing polycation. Further improvements in gene delivery were obtained by replacing polylysine with low molecular weight polyethylenimine (PEI) as the DNA condensing agent.
分子生物学有许多需要导入大（>100 kb）DNA分子的应用。目前大DNA转染的方法效率非常低。我们推断，用这些大DNA分子改进转染方法存在两个限制因素，即制备可行数量的纯净DNA存在困难，以及缺乏用于确定转染成功的快速检测方法。我们使用了基于大肠杆菌F因子质粒系统的细菌人工染色体（BAC），这种染色体易于操作且能以微克量进行纯化。由于BAC质粒在每个细胞中保持一到两个拷贝，消除了观察到的酵母人工染色体（YAC）的重排问题。我们构建了携带荧光素酶和绿色荧光蛋白（GFP）标记基因的两个系列的BAC载体。使用这些试剂，我们开发了将长达170 kb的BAC导入哺乳动物细胞的方法，其转染效率与5 - 10 kb DNA相当。补骨脂素灭活的腺病毒用作载体，从而消除了与病毒基因表达相关的问题。导入的DNA通过一种凝聚性聚阳离子与载体病毒相连。通过用低分子量聚乙烯亚胺（PEI）替代聚赖氨酸作为DNA凝聚剂，基因传递得到了进一步改进。
