title: Efficient intracellular delivery of native proteins.
abstract: 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.
authors.affiliation: KNAW-Hubrecht Institute, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands; University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands.
authors.forename: Diego S
authors.initials: DS
authors.lastname: D'Astolfo
authors.affiliation: University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands.
authors.forename: Romina J
authors.initials: RJ
authors.lastname: Pagliero
authors.affiliation: KNAW-Hubrecht Institute, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands.
authors.forename: Anita
authors.initials: A
authors.lastname: Pras
authors.affiliation: KNAW-Hubrecht Institute, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands; University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands.
authors.forename: Wouter R
authors.initials: WR
authors.lastname: Karthaus
authors.affiliation: KNAW-Hubrecht Institute, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands; University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands.
authors.forename: Hans
authors.initials: H
authors.lastname: Clevers
authors.affiliation: Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.
authors.forename: Vikram
authors.initials: V
authors.lastname: Prasad
authors.affiliation: University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands.
authors.forename: Robert Jan
authors.initials: RJ
authors.lastname: Lebbink
authors.affiliation: University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands.
authors.forename: Holger
authors.initials: H
authors.lastname: Rehmann
authors.affiliation: KNAW-Hubrecht Institute, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands; University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands; Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 108, 3584 CM Utrecht, the Netherlands. Electronic address: n.geijsen@hubrecht.eu.
authors.forename: Niels
authors.initials: N
authors.lastname: Geijsen
mesh_terms: D002478:Cells, Cultured; D003584:Cytological Techniques; D053595:Embryonic Stem Cells; D018390:Gene Targeting; D006801:Humans; D011506:Proteins; D012313:RNA; D014161:Transduction, Genetic
chemical_list: D011506:Proteins; D012313:RNA