Cargando…
Unexpected Transcellular Protein Crossover Occurs During Canonical DNA Transfection
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...
Autores principales: | , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BlackWell Publishing Ltd
2014
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263260/ https://www.ncbi.nlm.nih.gov/pubmed/25043607 http://dx.doi.org/10.1002/jcb.24884 |
_version_ | 1782348539662172160 |
---|---|
author | Arsenault, Jason Cuijpers, Sabine AG Niranjan, Dhevahi Davletov, Bazbek |
author_facet | Arsenault, Jason Cuijpers, Sabine AG Niranjan, Dhevahi Davletov, Bazbek |
author_sort | Arsenault, Jason |
collection | PubMed |
description | 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. J. Cell. Biochem. 115: 2047–2054, 2014. © 2014 Wiley Periodicals, Inc. |
format | Online Article Text |
id | pubmed-4263260 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | BlackWell Publishing Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-42632602014-12-15 Unexpected Transcellular Protein Crossover Occurs During Canonical DNA Transfection Arsenault, Jason Cuijpers, Sabine AG Niranjan, Dhevahi Davletov, Bazbek J Cell Biochem Benchmark 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. J. Cell. Biochem. 115: 2047–2054, 2014. © 2014 Wiley Periodicals, Inc. BlackWell Publishing Ltd 2014-12 2014-10-15 /pmc/articles/PMC4263260/ /pubmed/25043607 http://dx.doi.org/10.1002/jcb.24884 Text en © 2014 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc. http://creativecommons.org/licenses/by/3.0/ This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Benchmark Arsenault, Jason Cuijpers, Sabine AG Niranjan, Dhevahi Davletov, Bazbek Unexpected Transcellular Protein Crossover Occurs During Canonical DNA Transfection |
title | Unexpected Transcellular Protein Crossover Occurs During Canonical DNA Transfection |
title_full | Unexpected Transcellular Protein Crossover Occurs During Canonical DNA Transfection |
title_fullStr | Unexpected Transcellular Protein Crossover Occurs During Canonical DNA Transfection |
title_full_unstemmed | Unexpected Transcellular Protein Crossover Occurs During Canonical DNA Transfection |
title_short | Unexpected Transcellular Protein Crossover Occurs During Canonical DNA Transfection |
title_sort | unexpected transcellular protein crossover occurs during canonical dna transfection |
topic | Benchmark |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263260/ https://www.ncbi.nlm.nih.gov/pubmed/25043607 http://dx.doi.org/10.1002/jcb.24884 |
work_keys_str_mv | AT arsenaultjason unexpectedtranscellularproteincrossoveroccursduringcanonicaldnatransfection AT cuijperssabineag unexpectedtranscellularproteincrossoveroccursduringcanonicaldnatransfection AT niranjandhevahi unexpectedtranscellularproteincrossoveroccursduringcanonicaldnatransfection AT davletovbazbek unexpectedtranscellularproteincrossoveroccursduringcanonicaldnatransfection |