Zhao Xinxin, Su Huali, Yin Guangwen, Liu Xianyong, Liu Zhengzhu, Suo Xun
National Animal Protozoa Laboratory & College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
Vet Immunol Immunopathol. 2011 Dec 15;144(3-4):179-86. doi: 10.1016/j.vetimm.2011.10.003. Epub 2011 Oct 15.
Transgenic technology is an effective approach to assess the roles of specific genes in the activation and differentiation of T cells and modify T cell qualities. However, porcine T cell transfection is poorly documented. Here, we developed a non-virus-based method for the transfection of resting and ConA-stimulated porcine peripheral blood T cells using "Nucleofection" gene transfer technology; both plasmid DNA- and mRNA-mediated nucleofection systems were developed. The results demonstrated for the first time that plasmid DNA encoding green fluorescent protein (GFP) and in vitro transcribed GFP mRNA could be delivered efficiently into resting and activated porcine T cells. For both methods, the onset of gene expression was rapid and occurred within 2h post-nucleofection. Optimised plasmid DNA-mediated nucleofection induced approximately 40% transgene expression with 51% cell viability in resting T cells and approximately 20% transgene expression with 53% cell viability in activated T cells at 24h post-gene delivery. However, optimised mRNA-based nucleofection resulted in higher transfection efficiencies and cell viability, with more than 50% transgene expression and 62% viability for resting T cells and approximately 40% transgene expression and 59% viability for activated T cells. Finally, we measured the impact of the developed nucleofection systems on T cell function by detecting the mRNA levels of the activation markers CD25, CD69 and the cytokine IFN-γ; cell proliferation of the nucleofected resting peripheral blood mononuclear cells (PBMC) after ConA stimulation was also examined. The nucleofected resting PBMCs proliferated normally and up-regulated CD25, CD69 and IFN-γ mRNA expression levels in a manner comparable to non-nucleofected cells. These results indicate that the developed nucleofection systems have no adverse effects on T cell function and can be utilised in swine immunological research.
转基因技术是评估特定基因在T细胞激活和分化中的作用以及改善T细胞特性的有效方法。然而，猪T细胞转染的相关记录很少。在此，我们利用“核转染”基因转移技术开发了一种基于非病毒的方法，用于转染静止的和经刀豆蛋白A（ConA）刺激的猪外周血T细胞；同时开发了质粒DNA介导和mRNA介导的核转染系统。结果首次表明，编码绿色荧光蛋白（GFP）的质粒DNA和体外转录的GFP mRNA能够有效地导入静止的和活化的猪T细胞。对于这两种方法，基因表达开始迅速，在核转染后2小时内即可发生。优化后的质粒DNA介导的核转染在基因导入后24小时，可使静止T细胞诱导约40%的转基因表达，细胞活力为51%；使活化T细胞诱导约20%的转基因表达，细胞活力为53%。然而，优化后的基于mRNA的核转染产生了更高的转染效率和细胞活力，静止T细胞的转基因表达超过50%，细胞活力为62%；活化T细胞的转基因表达约为40%，细胞活力为59%。最后，我们通过检测激活标志物CD25、CD69和细胞因子IFN-γ的mRNA水平，来测定所开发的核转染系统对T细胞功能的影响；还检测了核转染后的静止外周血单个核细胞（PBMC）在ConA刺激后的细胞增殖情况。核转染后的静止PBMC正常增殖，并且以与未核转染细胞相当的方式上调CD25、CD69和IFN-γ mRNA表达水平。这些结果表明，所开发的核转染系统对T细胞功能没有不利影响，可用于猪的免疫学研究。
