Defining the EM-signature of successful cell-transfection.

In this report, we describe the architecture of Lipofectamine 2000 and 3000 transfection- reagents, as they appear inside of transfected cells, using classical transmission electron microscopy (EM). We also demonstrate that they provoke consistent structural changes after they have entered cells, changes that not only provide new insights into the mechanism of action of these particular transfection-reagents, but also provide a convenient and robust method for identifying by EM which cells in any culture have been successfully transfected. This also provides clues to the mechanism(s) of their toxic effects, when they are applied in excess. We demonstrate that after being bulk-endocytosed by cells, the cationic spheroids of Lipofectamine remain intact throughout the entire time of culturing, but escape from their endosomes and penetrate directly into the cytoplasm of the cell. In so doing, they provoke a stereotypical recruitment and rearrangement of endoplasmic reticulum (ER), and they ultimately end up escaping into the cytoplasm and forming unique 'inclusion-bodies.' Once free in the cytoplasm, they also invariably develop dense and uniform coatings of cytoplasmic ribosomes on their surfaces, and finally, they become surrounded by 'annulate' lamellae' of the ER. In the end, these annulate-lamellar enclosures become the ultrastructural 'signatures' of these inclusion-bodies, and serve to positively and definitively identify all cells that have been effectively transfected. Importantly, these new EM-observations define several new and unique properties of these classical Lipofectamines, and allow them to be discriminated from other lipoidal or particulate transfection-reagents, which we find do not physically break out of endosomes or end up in inclusion bodies, and in fact, provoke absolutely none of these 'signature' cytoplasmic reactions.