Ishino Takeshi, Kurita Hirofumi, Kirisawa Rikio, Shimamoto Yoshinori, Numano Rika, Kitamura Hiroshi
Laboratory of Veterinary Physiology, Departments of Veterinary Medicine, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan.
Department of Applied Chemistry and Life Sciences, Graduate School of Engineering, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan.
J Vet Med Sci. 2020 Jan 10;82(1):14-22. doi: 10.1292/jvms.19-0475. Epub 2019 Nov 27.
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.
仪器成本是将DNA片段和蛋白质导入细胞过程中的一个主要问题。油包水液滴电穿孔法（液滴电穿孔法）是最近发明的一种低成本且有效的将质粒转染到培养的人类细胞中的方法。我们在此将液滴电穿孔法应用于家畜动物细胞。尽管使用传统的脂质体转染方法很难将质粒转染到牛成纤维细胞中，但液滴电穿孔法使我们能够将表达增强型绿色荧光蛋白（EGFP）的质粒导入牛耳垂成纤维细胞。最佳转染条件为3.0 kV，此条件下19.1%的细胞被转染。对于猪耳垂成纤维细胞，在4.0 kV时最大转染效率为14.0%。用液滴电穿孔法转染后，69.1%的牛细胞和76.5%的猪细胞存活。此外，液滴电穿孔法在1.5 kV时成功地将大肠杆菌重组EGFP导入冻融后的牛精子中。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，液滴电穿孔法适用于将质粒和蛋白质转染到培养的家畜动物细胞中。
