Direct protein delivery to mammalian cells using cell-permeable Cys2-His2Zinc-finger domains

Thomas Gaj, Jia Liu

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article numbere52814
JournalJournal of Visualized Experiments
Volume2015
Issue number97
DOIs
StatePublished - Mar 25 2015

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Keywords

  • Cell-penetrating peptide
  • Chemical biology
  • Issue 97
  • Molecular biology
  • Molecular biology
  • Protein delivery
  • Protein transduction domain
  • Zinc-finger

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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