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Biochem Biophys Res Commun. 2016 May 20;474(1):8-14. doi: 10.1016/j.bbrc.2016.03.088. Epub 2016 Mar 21.

Single-cell real-time imaging of transgene expression upon lipofection.

Author information

1
Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy.
2
Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy; NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12, 56127, Pisa, Italy.
3
Department of Molecular Medicine, "Sapienza" University of Rome, Viale Regina Elena 291, 00161, Rome, Italy.
4
Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy. Electronic address: francesco.cardarelli@iit.it.

Abstract

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.

PMID:
27012199
DOI:
10.1016/j.bbrc.2016.03.088
[Indexed for MEDLINE]

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