Method for efficient transfection of in vitro-transcribed mRNA into SK-N-AS and HEK293 cells: difference in the toxicity of nuclear EGFP compared to cytoplasmic EGFP.
Method for efficient transfection of in vitro-transcribed mRNA into SK-N-AS and HEK293 cells: difference in the toxicity of nuclear EGFP compared to cytoplasmic EGFP.
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DOI：
10.3892/ijmm.17.6.1011
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发表时间：
2006-06
影响因子：
5.4
通讯作者：
K. Ejeskär;S. Fransson;F. Zaibak;P. Ioannou
中科院分区：
文献类型：
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作者：
K. Ejeskär;S. Fransson;F. Zaibak;P. Ioannou
在这里，我们报告了一种方法，有效地转染体外转录的mRNA到两种不同类型的人贴壁细胞，神经母细胞瘤细胞系SK-N-AS，和转化的肾细胞系HEK 293。用新的胰蛋白酶消化的贴壁细胞悬液和Lipofectaminetrademark 2000转染SK-N-AS和HEK 293细胞，转染效率分别为80-90%，转染后24 h，细胞活力分别为90%和60%。我们已经评估了主要定位于细胞质和细胞核的EGFP的不同作用，其中细胞核的EGFP比细胞质的EGFP对细胞的毒性更大。为了开发无效实验，我们构建了包括核定位信号的短的非功能性mRNA，并评估了可以添加编码核蛋白的mRNA而没有一般毒性的浓度，这取决于蛋白质定位于核的事实。对于SK-N-AS和HEK 293细胞，在10（5）个细胞中浓度高达100 ng的mRNA（编码无其他功能的核蛋白）不会影响细胞。为了评价该方法，我们筛选了四种不同的人mRNA，PDG，DFFA，CORT和PEX 14，以确定它们影响这些细胞中细胞增殖的能力。PEX 14是显著（p=0.03）降低两种细胞类型的细胞增殖的唯一基因，DFFA显著（p=0.04）降低SK-N-AS中的细胞增殖，但不降低HEK 293细胞中的细胞增殖。PGD和CORT对细胞增殖无影响。我们已经开发了一种简单的方法，用于将体外转录的mRNA有效地递送到贴壁细胞系SK-N-AS和HEK 293中。该方法可用于快速筛选不同基因如何影响细胞增殖。
Here we report a method for efficient transfection of in vitro-transcribed mRNA into two different types of human adherent cells, the neuroblastoma cell line SK-N-AS, and the transformed kidney cell line HEK293. By using newly trypsinized adherent cells in suspension and Lipofectaminetrade mark 2000, we detected a transfection efficiency of 80-90% in both cell lines and a cell viability of 90% in SK-N-AS and 60% in HEK293, 24 h after transfection when using cytoplasmic enhanced green fluorescent protein (EGFP)-mRNA. We have evaluated the different effects of the generally used EGFP that mainly localizes to the cytoplasm and nuclear EGFP, where the nuclear EGFP are more toxic to the cells than the cytoplasmic EGFP. In order to develop a null experiment, we constructed a short non-functional mRNA including a nuclear localization signal and evaluated the concentrations at which mRNA encoding nuclear proteins can be added without a general toxicity, depending on the fact that the proteins are localized to the nucleus. For both SK-N-AS and HEK293 cells, a concentration of up to 100 ng mRNA in 10(5) cells, encoding a nuclear protein with no other function, did not affect the cells. For evaluation of the method, we screened four different human mRNAs, PDG, DFFA, CORT and PEX14, for their ability to affect cell proliferation in these cells. PEX14 was the only gene that significantly (p=0.03) reduced cell proliferation for both cell types, DFFA significantly (p=0.04) reduced cell proliferation in SK-N-AS but not in HEK293 cells. PGD and CORT did not have any effect on cell proliferation. We have developed an easy method for efficient delivery of in vitro-transcribed mRNA into the adherent cell lines, SK-N-AS and HEK293. This method is useful for a quick screening of how different genes affect cell proliferation.