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Unexpected transcellular protein crossover occurs during canonical DNA transfection.

Arsenault J, Cuijpers SA, Niranjan D, Davletov B - J. Cell. Biochem. (2014)

Bottom Line: Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible.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.This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection.

View Article: PubMed Central - PubMed

Affiliation: MRC-Laboratory of Molecular Biology, Neurobiology Division, Cambridge, CB2 0QH, UK; Department of Pharmaceutical Sciences, University of Toronto, Toronto, Canada, M5S 3M2.

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Various modes of cellular entry of the type A protease (LcA) and immunoblotting of cleaved SNAP25. A: Western immunoblotting of total SNAP25 (SMI81) for Neuro2A cells treated with the type A protease under various conditions. Stressors such as sheering force or transfection reagents applied to the cells can mediate the entry of the protease. B–D: Confocal microscopy of cells immunostained with anti-cleaved SNAP25 antibody (red) and Hoechst stain (blue): (B) Control cells, (C) Mock-transfected cells, and (D) EGFP-LcA transfected cells (green). The anti-cleaved SNAP25 immunoreactivity can only be seen in cell populations subjected to LcA; this signal also extends beyond the detectable expression of the EGFP fluorophore.
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fig02: Various modes of cellular entry of the type A protease (LcA) and immunoblotting of cleaved SNAP25. A: Western immunoblotting of total SNAP25 (SMI81) for Neuro2A cells treated with the type A protease under various conditions. Stressors such as sheering force or transfection reagents applied to the cells can mediate the entry of the protease. B–D: Confocal microscopy of cells immunostained with anti-cleaved SNAP25 antibody (red) and Hoechst stain (blue): (B) Control cells, (C) Mock-transfected cells, and (D) EGFP-LcA transfected cells (green). The anti-cleaved SNAP25 immunoreactivity can only be seen in cell populations subjected to LcA; this signal also extends beyond the detectable expression of the EGFP fluorophore.

Mentions: As was previously shown [Kuo et al., 2010; Arsenault et al., 2014], transfection reagents can mediate the intracellular entry of BoNT holoenzymes. Figure 2A shows different conditions where unintended protease penetration can occur. Sheer force, representative of physical transfection methods, and different transfection reagents can cause the “transduction“ of the protease.


Unexpected transcellular protein crossover occurs during canonical DNA transfection.

Arsenault J, Cuijpers SA, Niranjan D, Davletov B - J. Cell. Biochem. (2014)

Various modes of cellular entry of the type A protease (LcA) and immunoblotting of cleaved SNAP25. A: Western immunoblotting of total SNAP25 (SMI81) for Neuro2A cells treated with the type A protease under various conditions. Stressors such as sheering force or transfection reagents applied to the cells can mediate the entry of the protease. B–D: Confocal microscopy of cells immunostained with anti-cleaved SNAP25 antibody (red) and Hoechst stain (blue): (B) Control cells, (C) Mock-transfected cells, and (D) EGFP-LcA transfected cells (green). The anti-cleaved SNAP25 immunoreactivity can only be seen in cell populations subjected to LcA; this signal also extends beyond the detectable expression of the EGFP fluorophore.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263260&req=5

fig02: Various modes of cellular entry of the type A protease (LcA) and immunoblotting of cleaved SNAP25. A: Western immunoblotting of total SNAP25 (SMI81) for Neuro2A cells treated with the type A protease under various conditions. Stressors such as sheering force or transfection reagents applied to the cells can mediate the entry of the protease. B–D: Confocal microscopy of cells immunostained with anti-cleaved SNAP25 antibody (red) and Hoechst stain (blue): (B) Control cells, (C) Mock-transfected cells, and (D) EGFP-LcA transfected cells (green). The anti-cleaved SNAP25 immunoreactivity can only be seen in cell populations subjected to LcA; this signal also extends beyond the detectable expression of the EGFP fluorophore.
Mentions: As was previously shown [Kuo et al., 2010; Arsenault et al., 2014], transfection reagents can mediate the intracellular entry of BoNT holoenzymes. Figure 2A shows different conditions where unintended protease penetration can occur. Sheer force, representative of physical transfection methods, and different transfection reagents can cause the “transduction“ of the protease.

Bottom Line: Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible.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.This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection.

View Article: PubMed Central - PubMed

Affiliation: MRC-Laboratory of Molecular Biology, Neurobiology Division, Cambridge, CB2 0QH, UK; Department of Pharmaceutical Sciences, University of Toronto, Toronto, Canada, M5S 3M2.

Show MeSH
Related in: MedlinePlus