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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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Measuring transfection yields. A: Flow cytometry analysis of Mock (gray) and EGFP-LcA (red) transfected Neuro2A cell distribution shown as a histogram of fluorescent intensity (FL1). B: Confocal microscopy image of EGFP-LcA transfected Neuro2A cells. White bar: 200 μm. C: Western immunoblotting of total SNAP25 (SMI81) showing the enzymatic activity of EGFP-LcA on transfected Neuro2A cells. A total conversion of SNAP25 can be observed. D: Calculated transfection percentage using the above three interpretation methodologies. There is a strong discrepancy between the fluorescently observed tranfection yields compared to total intracellular enzymatic activity.
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fig01: Measuring transfection yields. A: Flow cytometry analysis of Mock (gray) and EGFP-LcA (red) transfected Neuro2A cell distribution shown as a histogram of fluorescent intensity (FL1). B: Confocal microscopy image of EGFP-LcA transfected Neuro2A cells. White bar: 200 μm. C: Western immunoblotting of total SNAP25 (SMI81) showing the enzymatic activity of EGFP-LcA on transfected Neuro2A cells. A total conversion of SNAP25 can be observed. D: Calculated transfection percentage using the above three interpretation methodologies. There is a strong discrepancy between the fluorescently observed tranfection yields compared to total intracellular enzymatic activity.

Mentions: Following transient transfection, various methods can be used to determine the relative exogene expression yields. As can be seen in Figure 1, various traditional methods to quantify the expression of the transient exogene EGFP-LcA expression using Lipofectamine LTX into Neuro2A cells are presented. Using flow cytometry cells were gated by their forward and side-scatter (size and complexity) to select only morphologically normal cells. These gated cells were then quantified proportional to their EGFP fluorescent intensity (Fig. 1A). We also verified EGFP expression using confocal microscopy (Fig. 1B). Cells expressing the EGFP-LcA present a fluorescence that is generally localized around the cytosolic face of the plasma membrane. Western immunoblotting was also used to determine the total enzymatic effect within the entire sample. Total SNAP25 was immunoblotted using the SMI81 antibody revealing both the intact and the BoNT/A cleaved fragment. As can be seen in Figure 1C, the cleavage of the 9 C-terminal residues (SNAP25Δ9) leads to the appearance of a lower MW band. Surprisingly, this readout shows an improbably high transfection percentage. Figure 1D shows these quantifications where both flow cytometry and confocal microscopy correlates well despite the extra-ordinarily high enzymatic efficacy.


Unexpected transcellular protein crossover occurs during canonical DNA transfection.

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

Measuring transfection yields. A: Flow cytometry analysis of Mock (gray) and EGFP-LcA (red) transfected Neuro2A cell distribution shown as a histogram of fluorescent intensity (FL1). B: Confocal microscopy image of EGFP-LcA transfected Neuro2A cells. White bar: 200 μm. C: Western immunoblotting of total SNAP25 (SMI81) showing the enzymatic activity of EGFP-LcA on transfected Neuro2A cells. A total conversion of SNAP25 can be observed. D: Calculated transfection percentage using the above three interpretation methodologies. There is a strong discrepancy between the fluorescently observed tranfection yields compared to total intracellular enzymatic activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig01: Measuring transfection yields. A: Flow cytometry analysis of Mock (gray) and EGFP-LcA (red) transfected Neuro2A cell distribution shown as a histogram of fluorescent intensity (FL1). B: Confocal microscopy image of EGFP-LcA transfected Neuro2A cells. White bar: 200 μm. C: Western immunoblotting of total SNAP25 (SMI81) showing the enzymatic activity of EGFP-LcA on transfected Neuro2A cells. A total conversion of SNAP25 can be observed. D: Calculated transfection percentage using the above three interpretation methodologies. There is a strong discrepancy between the fluorescently observed tranfection yields compared to total intracellular enzymatic activity.
Mentions: Following transient transfection, various methods can be used to determine the relative exogene expression yields. As can be seen in Figure 1, various traditional methods to quantify the expression of the transient exogene EGFP-LcA expression using Lipofectamine LTX into Neuro2A cells are presented. Using flow cytometry cells were gated by their forward and side-scatter (size and complexity) to select only morphologically normal cells. These gated cells were then quantified proportional to their EGFP fluorescent intensity (Fig. 1A). We also verified EGFP expression using confocal microscopy (Fig. 1B). Cells expressing the EGFP-LcA present a fluorescence that is generally localized around the cytosolic face of the plasma membrane. Western immunoblotting was also used to determine the total enzymatic effect within the entire sample. Total SNAP25 was immunoblotted using the SMI81 antibody revealing both the intact and the BoNT/A cleaved fragment. As can be seen in Figure 1C, the cleavage of the 9 C-terminal residues (SNAP25Δ9) leads to the appearance of a lower MW band. Surprisingly, this readout shows an improbably high transfection percentage. Figure 1D shows these quantifications where both flow cytometry and confocal microscopy correlates well despite the extra-ordinarily high enzymatic efficacy.

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