Reversible Membrane Permeabilization of Mammalian Cells Treated with Digitonin and Its Use for Inducing Nuclear Reprogramming by Xenopus Egg Extracts
Reversible Membrane Permeabilization of Mammalian Cells Treated with Digitonin and Its Use for Inducing Nuclear Reprogramming by Xenopus Egg Extracts
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DOI：
10.1089/clo.2008.0020
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发表时间：
2008-12-01
期刊：
影响因子：
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通讯作者：
Imai, Hiroshi
中科院分区：
文献类型：
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作者：
Miyamoto, Kei;Yamashita, Teruyoshi;Imai, Hiroshi
质膜可被链球菌溶血素O可逆地透化。透化细胞在卵提取物的无细胞体系中可发生重编程和部分去分化。然而，链球菌溶血素0的透化活性不稳定，因此难以控制其活性。可逆透化的替代方法可用于建立无细胞系统。在这里，我们使用了非离子洗涤剂，毛地黄皂苷，渗透。低浓度毛地黄皂苷诱导牛、小鼠和猪体细胞质膜可逆透化。用Xenoptis laevis卵提取物处理透化的细胞。处理后的细胞显示提取物中的核蛋白发生交换，例如Xenopus特异性组蛋白B4和核纤层蛋白LIII掺入细胞核中。重新密封膜后，细胞显示OCT 4、SOX 2和NANOG表达上调。我们的研究结果表明，可逆透化毛地黄皂苷可用于诱导核重编程和激活多能基因的无细胞系统。
Plasma membranes can be reversibly permeabilized by Streptolysin O. The permeabilized cells can be reprogrammed and partially dedifferentiated in the cell-free system from egg extracts. However, the permeabilizing activity of Streptolysin 0 is not stable, and therefore it is difficult to control its activity. An alternative method for reversible permeabilization is useful for establishing a cell-free system. Here, we used a nonionic detergent, digitonin, for permeabilization. A low concentration of digitonin induced reversible permeabilization of the plasma membrane in bovine, mouse, and porcine somatic cells. The permeabilized cells were treated with Xenoptis laevis egg extracts. The treated cells showed exchange of nuclear proteins from extracts such as incorporation of Xenopus-specific histone B4 and Lamin LIII into nuclei. After resealing of the membrane, the cells showed upregulation of OCT4, SOX2, and NANOG expression. Our results suggest that reversible permeabilization with digitonin can be used to induce nuclear reprogramming and to activate pluripotent genes by a cell-free system.