title: Single-cell real-time imaging of transgene expression upon lipofection.
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.
authors.affiliation: Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Fiume
authors.affiliation: 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.
authors.forename: Carmine
authors.initials: C
authors.lastname: Di Rienzo
authors.affiliation: Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Marchetti
authors.affiliation: Department of Molecular Medicine, "Sapienza" University of Rome, Viale Regina Elena 291, 00161, Rome, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Pozzi
authors.affiliation: Department of Molecular Medicine, "Sapienza" University of Rome, Viale Regina Elena 291, 00161, Rome, Italy.
authors.forename: Giulio
authors.initials: G
authors.lastname: Caracciolo
authors.affiliation: Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy. Electronic address: francesco.cardarelli@iit.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Cardarelli
mesh_terms: D000818:Animals; D016466:CHO Cells; D003199:Computer Systems; D003412:Cricetulus; D020869:Gene Expression Profiling / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D008055:Lipids / Q000737:chemistry*; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014162:Transfection / Q000379:methods*; D019076:Transgenes / Q000235:genetics*
chemical_list: D008055:Lipids; C086724:Lipofectamine; D049452:Green Fluorescent Proteins