Efficient DNA transfection in neuronal and astrocytic cell lines
Efficient DNA transfection in neuronal and astrocytic cell lines
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DOI：
10.1023/a:1007173906990
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发表时间：
2000-06-01
影响因子：
2.8
通讯作者：
Lahiri, DK
中科院分区：
文献类型：
--
作者：
Ghosh, C;Song, W;Lahiri, DK
我们研究了不同的参数在不同的细胞类型和不同大小的启动子有效的DNA转染。本研究报告了PC12细胞电穿孔DNA转染的最佳条件为350 V/960 muF， C6细胞为450V/960 muF， COS-1细胞为250 V/500 muF。对于人神经母细胞瘤（SK-N-SH）细胞，最佳的DNA转染条件是磷酸钙法。在启动子图谱研究中，常用的是序列删除方法。为了优化转染，我们选择了三个大小从4.5到12.4千碱基（kb）不等的DNA构建体。我们在“等量”、“等摩尔”和“等摩尔加载体DNA使其等量”的条件下测量了这些结构体的启动子活性。为了便于比较，我们建议在“等摩尔条件”下进行转染，而不需要通过载体DNA调整DNA总量。综上所述，我们的研究结果表明，通过设计更好的方法将DNA传递到哺乳动物细胞中，有效的DNA转染方法对于研究基因调控非常重要。
We have studied different parameters for efficient DNA transfection in various cell types and with different size of the promoter. Here we report that the optimum condition for DNA transfection by electroporation is 350 V/960 muF for PC12, 450V/960 muF C6 cells, and 250 V/500 muF for COS-1 cells. For the human neuroblastoma (SK-N-SH) cells the optimum condition for DNA transfection is by the calcium phosphate method. In promoter mapping studies, a serial deletion approach is commonly used. To optimize transfection we have selected three DNA constructs that varied in size from 4.5 to 12.4 kilobases (kb). We measured the promoter activity of these constructs under conditions of 'equal amount', 'equimolar', and 'equimolar plus carrier DNA to make it equal amount'. We recommend that for comparative purpose, transfection should be carried out under 'equimolar condition' without a need to adjust the total amount of DNA by carrier DNA. Taken together, our results suggest that efficient methods for DNA transfection are important to study gene regulation by devising better ways to deliver DNA into the mammalian cells.