title: Direct protein delivery to mammalian cells using cell-permeable Cys2-His2 zinc-finger domains.
abstract: Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.
authors: Departments of Chemistry and Cell and Molecular Biology, The Scripps Research Institute; gaj@berkeley.edu.
authors: Thomas
authors: T
authors: Gaj
authors: Departments of Chemistry and Cell and Molecular Biology, The Scripps Research Institute; Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University.
authors: Jia
authors: J
authors: Liu
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002463:Cell Membrane Permeability; D003545:Cysteine / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000008:administration & dosage / Q000737:chemistry; D006367:HeLa Cells; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011506:Proteins / Q000008:administration & dosage* / Q000737:chemistry; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; D011506:Proteins; D006639:Histidine; D003545:Cysteine