Fiume Giuseppe, Di Rienzo Carmine, Marchetti Laura, Pozzi Daniela, Caracciolo Giulio, Cardarelli Francesco
Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy.
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.
Biochem Biophys Res Commun. 2016 May 20;474(1):8-14. doi: 10.1016/j.bbrc.2016.03.088. Epub 2016 Mar 21.
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.
在这里，我们通过在单细胞水平上对基因编码荧光报告基因的表达进行定量，并在活细胞中通过共聚焦成像进行实时定量，来研究脂质转染过程。将脂质体金标准制剂与有前景的替代制剂DC-Chol/DOPE进行比较。在这两种情况下，我们都报告说只有分裂细胞能够产生可检测量的荧光报告蛋白。值得注意的是，通过测量每对子细胞随时间的荧光，我们发现与DC-Chol/DOPE相比，基于脂质体的转染在子细胞之间的蛋白质表达上统计学上产生了显著更高程度的“对称性”。设想了一个模型，其中蛋白质表达的对称程度与核破裂前细胞内生物可利用DNA拷贝数相关。报道的结果为理解脂质转染机制开辟了新的视角，并定义了一个用于转染试剂定量比较的新实验平台。
