title: Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT.
abstract: Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.
authors.affiliation: Biochemistry and Biophysics, Texas A&M University.
authors.forename: Kristina
authors.initials: K
authors.lastname: Najjar
authors.affiliation: Biochemistry and Biophysics, Texas A&M University.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Erazo-Oliveras
authors.affiliation: Biochemistry and Biophysics, Texas A&M University; Pellois@tamu.edu.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Pellois
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D057846:Cell-Penetrating Peptides / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D016503:Drug Delivery Systems / Q000379:methods*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005456:Fluorescent Dyes / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics; D010455:Peptides / Q000008:administration & dosage* / Q000493:pharmacokinetics; D011108:Polymers / Q000737:chemistry; D011506:Proteins / Q000008:administration & dosage* / Q000493:pharmacokinetics; D012235:Rhodamines / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics
chemical_list: D057846:Cell-Penetrating Peptides; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D010455:Peptides; D011108:Polymers; D011506:Proteins; D012235:Rhodamines; C005358:tetramethylrhodamine