Decastro Michael, Saijoh Yukio, Schoenwolf Gary C
Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.
Dev Dyn. 2006 Aug;235(8):2210-9. doi: 10.1002/dvdy.20873.
We have used cationic lipid-based transfection reagents for ectopic gene expression experiments in developing vertebrate embryos. Lipofectamine, Lipofectamine 2000, and Lipofectamine enhanced with a disulfide linked pegylated lipid (mPEG-SS-DOPE) were initially tested and optimized in cell culture. Two reagent formulations, 1:4 (DNA:Lipofectamine 2000) Lipofectamine 2000, and 7.5% pegylated Lipofectamine, produced the highest levels of gene expression in vitro. Those formulations, containing the enhance green fluorescent protein reporter gene, were microinjected into intact vertebrate embryos -- systemically through the vasculature and locally into selected tissues -- to assess in vivo transfection efficiency. Whereas both formulations are capable of transfecting cells in developing embryos in vivo, greater transfection efficiencies in a broader range of tissue types were obtained with the pegylated Lipofectamine formulation. We conclude that in developing vertebrate embryos, optimized cationic lipid-based reagents are capable of producing significant levels of ectopic gene expression and can be used as alternatives to electroporation and viral-mediated gene delivery.
我们已将基于阳离子脂质的转染试剂用于发育中的脊椎动物胚胎的异位基因表达实验。最初在细胞培养中对脂质体转染试剂、脂质体2000以及用二硫键连接的聚乙二醇化脂质（mPEG-SS-DOPE）增强的脂质体转染试剂进行了测试和优化。两种试剂配方，即1:4（DNA:脂质体2000）的脂质体2000和7.5%的聚乙二醇化脂质体转染试剂，在体外产生了最高水平的基因表达。将含有增强型绿色荧光蛋白报告基因的那些配方通过血管系统全身以及局部注射到选定组织中，注入完整的脊椎动物胚胎，以评估体内转染效率。虽然两种配方都能够在体内发育中的胚胎中转染细胞，但聚乙二醇化脂质体转染试剂配方在更广泛的组织类型中获得了更高的转染效率。我们得出结论，在发育中的脊椎动物胚胎中，优化的基于阳离子脂质的试剂能够产生显著水平的异位基因表达，并且可以用作电穿孔和病毒介导的基因递送的替代方法。
