Stem Cell Engineering Group, Institute of Reconstructive Neurobiology, University of Bonn - Life & Brain Center and Hertie Foundation, Sigmund-Freud Straße 25, 53105 Bonn, Germany.
Methods. 2011 Apr;53(4):386-93. doi: 10.1016/j.ymeth.2010.12.018. Epub 2010 Dec 23.
Protein transduction is based on the ability of certain peptides, designated as cell penetrating peptides (CPPs), to intracellularly deliver cargo molecules, such as peptides and proteins. In combination with site specific recombination, CPP-mediated delivery of recombinases enables a precise and highly efficient control of gene expression in cultured cells and mice. Herein, we provide detailed protocols for engineering and purification of a cell-permeant FLP recombinase protein. Two examples describe the use of cell permeant FLP for excising prespecified fragments from transgenes expressed in fibroblasts and mouse embryonic stem cells. A third example describes the combined use of cell-permeant Cre and FLP recombinases to reversibly induce transgenes in embryonic stem cells. We anticipate that the protocols described herein will be widely used for various genetic interventions addressing complex biological questions.
蛋白质转导基于某些肽的能力，这些肽被指定为细胞穿透肽（CPP），可以将货物分子（如肽和蛋白质）递送到细胞内。与位点特异性重组结合，CPP 介导的重组酶递送使在培养细胞和小鼠中精确且高效地控制基因表达成为可能。在此，我们提供了用于工程化和纯化细胞穿透性 FLP 重组酶蛋白的详细方案。两个例子描述了使用细胞穿透性 FLP 从成纤维细胞和小鼠胚胎干细胞中表达的转基因中切除预定片段的用途。第三个例子描述了使用细胞穿透性 Cre 和 FLP 重组酶可逆诱导胚胎干细胞中转基因的联合使用。我们预计，本文描述的方案将广泛用于解决复杂生物学问题的各种遗传干预。
