Shi Hong, Ruan Lingwei, Söderhäll Irene, Söderhäll Kenneth, Xu Xun
State Key Laboratory Breeding Base of Marine Genetic Resources, Key Laboratory of Marine Genetic Resources of State Oceanic Administration, Third Institute of Oceanography, State Oceanic Administration, Fujian Key Laboratory of Marine Genetic Resources, South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Xiamen, 361005, PR China.
State Key Laboratory Breeding Base of Marine Genetic Resources, Key Laboratory of Marine Genetic Resources of State Oceanic Administration, Third Institute of Oceanography, State Oceanic Administration, Fujian Key Laboratory of Marine Genetic Resources, South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Xiamen, 361005, PR China.
Dev Comp Immunol. 2018 Nov;88:70-76. doi: 10.1016/j.dci.2018.07.005. Epub 2018 Jul 9.
Transfection is a powerful tool useful for studying gene function. Establishing transfection methods that enable highly efficient DNA uptake has become increasingly important. The crayfish hematopoietic tissue (Hpt) cell cultures have been proven to be suitable for studies on immunity and cell differentiation in crustaceans including shrimps, but no efficient gene transfer and expression method is available for these cells. Here we report a novel and highly efficient DNA transfection system based on electroporation. This method depends on a recombinant plasmid with the promoter from white spot syndrome virus immediate-early gene wsv249. This plasmid could be introduced into primary cells and efficiently express foreign genes by electroporation. By optimizing different electroporation parameters, more than 30% transfection efficiency could be achieved with the relative viability of cells around 50%. This is the first report of gene introduction to crayfish Hpt cells and will be useful for the expanding our research on crustacean immunity.
转染是研究基因功能的一种强大工具。建立能够实现高效DNA摄取的转染方法变得越来越重要。小龙虾造血组织（Hpt）细胞培养已被证明适用于包括虾类在内的甲壳类动物的免疫和细胞分化研究，但这些细胞尚无有效的基因转移和表达方法。在此，我们报告一种基于电穿孔的新型高效DNA转染系统。该方法依赖于一种带有白斑综合征病毒立即早期基因wsv249启动子的重组质粒。通过电穿孔，该质粒可导入原代细胞并有效表达外源基因。通过优化不同的电穿孔参数，可实现超过30%的转染效率，细胞相对活力约为50%。这是关于将基因导入小龙虾Hpt细胞的首次报道，将有助于拓展我们对甲壳类动物免疫的研究。
