Transfection, referring to the 'infection of a cell with free nucleic acid' [1], is a powerful and widely used technique for manipulating the expression of proteins in mammalian cells. The technique of transfection has since been modified from its original meaning and now also encompasses techniques utilizing shuttling agents to carry nucleic acids across the plasma membrane, including viral vectors, naked plasmid DNA, and cationic liposome complexes of plasmid DNA. It is often assumed that the protein of interest can be expressed without significant adverse effects from the transfection technique itself. Indeed, this is the case with many transfection strategies and this facilitates the study of many proteins in situ within cells. Often little consideration is given to how the expression of the transgene affects the biology of the cell outside of the parameters being followed.
In this issue of the journal, de Carvalho Bittencourt et al. examine the effect of transfection with naked DNA, liposome-complexed DNA, and retroviral vectors on the human retrovirus packaging cell line Y-CRIP. Using this cell line, which is resistant to Fas-signaled apoptosis, these authors demonstrate that transfection with plasmid DNA increases susceptibility to apoptotic signals delivered by recombinant FasL and induces the expression of the costimulatory molecule CD80. Further, these authors demonstrate that the increased susceptibility occurs regardless of the protein encoded by the plasmid used for transfection. In contrast, transfection with a retroviral vector encoding the same insert did not affect the susceptibility to recombinant FasL signaling.
These results raise some interesting questions. Foremost, is transfection with a retroviral vector (receptor-mediated) a different and perhaps more innocuous process than transfection with plasmid DNA? Of course, transfection through a retroviral vector is a more complicated process by virtue of the infection cycle of the retrovirus. Yet these results suggest that the pathology associated with the transfection processes is minimal and did not adversely affect the cell. There are at least two key differences that may a priori make these two methods of transfection different from the point of view of the cell and may account for the differences observed. First, retroviral transfection is associated with integration of the proviral DNA and the transgene sequence encoding the insert. Plasmid DNA, however, is believed to remain episomal for the most part following transfection. Additionally, retroviruses utilize receptor-mediated entry using host cell proteins to gain entry into the cell, e.g. CD4 and chemokine receptors in the case of HIV. Plasmid DNA transfection is thought to be a random event; however, recently it has been shown that mammalian cells can recognize the presence of bacterial DNA sequences through the Toll-like receptor-9 (TLR-9) [2].
The interaction of plasmid DNA with the TLR-9 is a significant difference between transfection with plasmids and retroviral vectors. This is particularly important from the standpoint of activation of the immune system, as DNA transfection would be associated with rapid activation of the immune response. The results of these studies suggest that retroviral transfection methods would probably be advantageous in the delivery of genes for 'gene