KNAW-Hubrecht Institute, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands; University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands.
University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands.
Cell. 2015 Apr 23;161(3):674-690. doi: 10.1016/j.cell.2015.03.028.
Modulation of protein function is used to intervene in cellular processes but is often done indirectly by means of introducing DNA or mRNA encoding the effector protein. Thus far, direct intracellular delivery of proteins has remained challenging. We developed a method termed iTOP, for induced transduction by osmocytosis and propanebetaine, in which a combination of NaCl hypertonicity-induced macropinocytosis and a transduction compound (propanebetaine) induces the highly efficient transduction of proteins into a wide variety of primary cells. We demonstrate that iTOP is a useful tool in systems in which transient cell manipulation drives permanent cellular changes. As an example, we demonstrate that iTOP can mediate the delivery of recombinant Cas9 protein and short guide RNA, driving efficient gene targeting in a non-integrative manner.
蛋白质功能的调节被用来干预细胞过程，但通常是通过引入编码效应蛋白的 DNA 或 mRNA 来间接实现。到目前为止，蛋白质的直接细胞内递送仍然具有挑战性。我们开发了一种称为 iTOP 的方法，用于渗透压和丙基甜菜碱诱导转导，其中 NaCl 高渗诱导的巨胞饮作用和转导化合物（丙基甜菜碱）的组合诱导蛋白质高效转导到各种原代细胞中。我们证明，iTOP 是一种在瞬时细胞操作驱动永久细胞变化的系统中非常有用的工具。例如，我们证明 iTOP 可以介导重组 Cas9 蛋白和短向导 RNA 的递送，以非整合的方式有效地进行基因靶向。
