Introduction of a plasmid and a protein into bovine and swine cells by water-in-oil droplet electroporation
Introduction of a plasmid and a protein into bovine and swine cells by water-in-oil droplet electroporation
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DOI：
10.1292/jvms.19-0475
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发表时间：
2020-01-01
影响因子：
1.2
通讯作者：
Kitamura, Hiroshi
中科院分区：
文献类型：
--
作者：
Ishino, Takeshi;Kurita, Hirofumi;Kitamura, Hiroshi
仪器成本是将 DNA 片段和蛋白质转导到细胞中的一个主要问题。油包水液滴电穿孔（droplet-EP）是最近发明的一种低成本且有效的将质粒转染至培养的人类细胞中的方法。我们在这里将 Droplet-EP 应用于家畜动物细胞。尽管使用传统的脂转染方法很难将质粒转染到牛成纤维细胞中，但液滴-EP使我们能够将表达增强型绿色荧光蛋白（EGFP）的质粒引入牛耳垂成纤维细胞中。最佳转染条件为3.0 kV，可转染19.1%的细胞。对于猪耳垂成纤维细胞，4.0 kV 时的最大转染效率为 14.0%。用 Droplet-EP 转染后，69.1% 的牛细胞和 76.5% 的猪细胞存活。此外，droplet-EP 在 1.5 kV 下成功将大肠杆菌重组 EGFP 转导至冻融的牛精子中。流式细胞术分析显示，转染后71.5%的精子呈现绿色荧光。总体而言，droplet-EP适合将质粒和蛋白质转染至培养的家畜动物细胞中。
Instrument cost is a major problem for the transduction of DNA fragments and proteins into cells. Water-in-oil droplet electroporation (droplet-EP) was recently invented as a low-cost and effective method for the transfection of plasmids into cultured human cells. We here applied droplet-EP to livestock animal cells. Although it is difficult to transfect plasmids into bovine fibroblasts using conventional lipofection methods, droplet-EP enabled us to introduce an enhanced green fluorescent protein (EGFP)-expressing plasmid into bovine earlobe fibroblasts. The optimal transfection condition was 3.0 kV, which allowed 19.1% of the cells to be transfected. For swine earlobe fibroblasts, the maximum transfection efficacy was 14.0% at 4.0 kV. After transfection with droplet-EP, 69.1% of bovine and 76.5% of swine cells were viable. Furthermore, droplet-EP successfully transduced Escherichia coli recombinant EGFP into frozen-thawed bovine sperm at 1.5 kV. Flow cytometry analysis revealed that 71.5% of spermatozoa exhibited green fluorescence after transfection. Overall, droplet-EP is suitable for the transfection of plasmids and proteins into cultured livestock animal cells.