title: High transfection efficiency of porcine peripheral blood T cells via nucleofection.
abstract: 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.
authors: National Animal Protozoa Laboratory & College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
authors: Xinxin
authors: X
authors: Zhao
authors: Huali
authors: H
authors: Su
authors: Guangwen
authors: G
authors: Yin
authors: Xianyong
authors: X
authors: Liu
authors: Zhengzhu
authors: Z
authors: Liu
authors: Xun
authors: X
authors: Suo
mesh_terms: D000818:Animals; D002470:Cell Survival; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018274:Electroporation / Q000379:methods*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D013552:Swine; D013601:T-Lymphocytes / Q000378:metabolism*; D013778:Tetrazolium Salts; D013844:Thiazoles; D014162:Transfection / Q000379:methods*
chemical_list: D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013844:Thiazoles; D049452:Green Fluorescent Proteins; D004247:DNA; C022616:thiazolyl blue