Dennaya Kumara, Hayfa Salsabila Harsan, Metta Novianti, Dinda Lestari, E. P. Septisetyani, P. W. Prasetyaningrum, K. A. Paramitasari, Edy Meiyanto
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This plasmid is helpful as a biosensor of the cytoskeleton and nucleus that enables live imaging observation using a fluorescence microscope, for instance, in the observation of syncytium. Here, we optimized two independent variables: the amount of DNA (0.5 and 1 µg) and the ratio of DNA-PEI (1:3 and 1:4). GFP and mCherry expressions were observed at 24, 48, and 72 h post-transfection. As a result, transfection efficiency achieved by using PEI in 293T cells is higher than in BHK-21 cells, which are ~90% and ~50%, respectively. Moreover, amongst four different transfection conditions, in both cell lines, 1 µg of plasmid DNA with a 1:3 DNA-PEI ratio yields the most efficiency with the least amount of toxicity. We used this condition for the syncytia observation in 293T cells as a model of the cell-to-cell transmission of SARS-CoV-2. 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引用次数: 0
摘要
转染效率对质粒 DNA 能否成功转入细胞起着积极作用,重点在于质粒 DNA 的数量及其与转染试剂的比例。聚乙烯亚胺(PEI)是一种经济有效的转染试剂,它通过形成带正电荷的 DNA 复合物来促进 DNA 的转移。它能使 DNA 与带负电荷的细胞表面相互作用,并通过内吞作用进入细胞。在本研究中,我们优化了使用 PEI 在 BHK-21 和 293T 细胞中瞬时转染表达生命 act-GFP/NLS-mCherry 质粒的条件。该质粒可作为细胞骨架和细胞核的生物传感器,使用荧光显微镜进行活体成像观察,例如观察合胞体。在这里,我们优化了两个自变量:DNA 的用量(0.5 和 1 µg)和 DNA-PEI 的比例(1:3 和 1:4)。在转染后 24、48 和 72 小时观察到了 GFP 和 mCherry 的表达。因此,在 293T 细胞中使用 PEI 实现的转染效率高于 BHK-21 细胞,分别为 ~90% 和 ~50%。此外,在这两种细胞系的四种不同转染条件中,1 µg 的质粒 DNA 与 1:3 的 DNA-PEI 比率产生的转染效率最高,毒性最小。我们使用这种条件在 293T 细胞中观察合胞体,以此作为 SARS-CoV-2 细胞间传播的模型。通过检测表达多核 GFP/mCherry 的巨细胞,我们成功地观察到了合胞体的形成。
Optimized condition for pei-based transient transfection of lifeact-gfp/nls-mcherry expressing plasmid used as cell barcode for syncytia live cell imaging
The transfection efficiency positively affects the successful plasmid DNA transfer into cells, with the highlight on the amount of plasmid DNA and its ratio to the transfection reagent. Polyethyleneimine (PEI) is a cost-effective transfection reagent that facilitates DNA transfer by forming positively charged DNA complexes. It allows DNA to interact with negatively charged cell surfaces and enter the cells by endocytosis. In this study, we optimized the condition for transient transfection of life act-GFP/NLS-mCherry-expressing plasmid in BHK-21 and 293T cells using PEI. This plasmid is helpful as a biosensor of the cytoskeleton and nucleus that enables live imaging observation using a fluorescence microscope, for instance, in the observation of syncytium. Here, we optimized two independent variables: the amount of DNA (0.5 and 1 µg) and the ratio of DNA-PEI (1:3 and 1:4). GFP and mCherry expressions were observed at 24, 48, and 72 h post-transfection. As a result, transfection efficiency achieved by using PEI in 293T cells is higher than in BHK-21 cells, which are ~90% and ~50%, respectively. Moreover, amongst four different transfection conditions, in both cell lines, 1 µg of plasmid DNA with a 1:3 DNA-PEI ratio yields the most efficiency with the least amount of toxicity. We used this condition for the syncytia observation in 293T cells as a model of the cell-to-cell transmission of SARS-CoV-2. Syncytia formation was successfully observed by detecting the giant cells expressing GFP/mCherry with multiple nuclei.