title: Unexpected transcellular protein crossover occurs during canonical DNA transfection.
abstract: Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible. Here, we demonstrate that Neuro2A cells transfected using Lipofectamine LTX with the fluorescently coupled Botulinum serotype A holoenzyme (EGFP-LcA) cDNA express this SNAP25 protease that can, once translated, escape the transfected host cytosol and become endocytosed into untransfected cells, without its innate binding and translocation domains. Fluorescent readouts revealed moderate transfection rates (30-50%) while immunoblotting revealed a surprisingly total enzymatic cleavage of SNAP25; the transgenic protein acted beyond the confines of its host cell. Using intracellular dyes, no important cytotoxic effects were observed from reagent treatment alone, which excluded the possibility of membrane ruptures, though noticeably, intracellular acidic organelles were redistributed towards the plasma membrane. This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection.
authors.affiliation: MRC-Laboratory of Molecular Biology, Neurobiology Division, Cambridge, CB2 0QH, UK; Department of Pharmaceutical Sciences, University of Toronto, Toronto, Canada, M5S 3M2.
authors.forename: Jason
authors.initials: J
authors.lastname: Arsenault
authors.forename: Sabine A G
authors.initials: SA
authors.lastname: Cuijpers
authors.forename: Dhevahi
authors.initials: D
authors.lastname: Niranjan
authors.forename: Bazbek
authors.initials: B
authors.lastname: Davletov
mesh_terms: D000818:Animals; D019274:Botulinum Toxins, Type A / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004705:Endocytosis; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008055:Lipids / Q000494:pharmacology; D051379:Mice; D015388:Organelles; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D050825:Synaptosomal-Associated Protein 25 / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
chemical_list: D008055:Lipids; C086724:Lipofectamine; D011993:Recombinant Fusion Proteins; D050825:Synaptosomal-Associated Protein 25; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019274:Botulinum Toxins, Type A; C545476:incobotulinumtoxinA
keywords: BOTULINUM NEUROTOXIN; ENDOCYTOSIS; INTERNALIZATION; LIPOFECTION; SNARE; TRAFFICKING; TRANSFECTION