Here we address the process of lipofection by quantifying the expression of a genetically-encoded fluorescent reporter at the single-cell level, and in real-time, by confocal imaging in live cells. The Lipofectamine gold-standard formulation is compared to the alternative promising DC-Chol/DOPE formulation. In both cases, we report that only dividing cells are able to produce a detectable amount of the fluorescent reporter protein. Notably, by measuring fluorescence over time in each pair of daughter cells, we find that Lipofectamine-based transfection statistically yields a remarkably higher degree of "symmetry" in protein expression between daughter cells as compared to DC-Chol/DOPE. A model is envisioned in which the degree of symmetry of protein expression is linked to the number of bioavailable DNA copies within the cell before nuclear breakdown. Reported results open new perspectives for the understanding of the lipofection mechanism and define a new experimental platform for the quantitative comparison of transfection reagents.
|Titolo:||Single-cell real-time imaging of transgene expression upon lipofection|
|Data di pubblicazione:||2016|
|Parole Chiave:||Animals; CHO Cells; Computer Systems; Cricetulus; Gene Expression Profiling; Genes, Reporter; Green Fluorescent Proteins; Lipids; Microscopy, Confocal; Microscopy, Fluorescence; Molecular Imaging; Reproducibility of Results; Sensitivity and Specificity; Transfection; Transgenes; Biophysics; Biochemistry; Molecular Biology; Cell Biology|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1016/j.bbrc.2016.03.088|
|Appare nelle tipologie:||1.1 Articolo in rivista|