We read with interest the article by Maucksch et al. [1], who analysed transgene expression of transfected supercoiled 4.7 kb monomeric and 9.4 kb dimeric plasmid concatemer in Jurkat T cells using electroporation. The authors reported that the relative number of electrotransferred gene copies per nucleus and plasmid expression efficiency, respectively, were 1.6-and 3.5-fold higher for enhanced green fluorescent protein (EGFP)-dimer than for EGFP-monomer. However, the transfection rates considering the number of transfected cells were significantly lower for EGFP-dimer than for EGFPmonomer. Their conclusions were that more dimers were introduced in fewer cells.
The relative number of gene copies introduced in the different cellular compartments (cytoplasm and nucleus) was evaluated using a fluorescence approach, which we described some years ago [2]. This took advantage of the spontaneous binding of TOTO dyes to DNA.
Therefore, Maucksch et al.
[1] have used noncovalent markers (SYTO-13 and TOTO-1) to determine the biodistribution of plasmids into the cells after electroporation. TOTO is virtually nonfluorescent in solution, but forms highly fluorescent complexes with double-stranded DNA (dsDNA), up to a maximum dye to DNA bp ratio of 1 : 4, with a 1000-fold fluorescence enhancement. The dsDNA-TOTO complexes are completely stable to electrophoresis on agarose and acrylamide gels [3]. However, TOTO-1 is a dye capable of bisintercalation, although it interacts with dsDNA and ssDNA with a similar high affinity. Binding of the dye partially unwinds the DNA by distorting and elongating the helix. An external binding mode, where the dipole of the dye molecule is aligned with the DNA grooves, may be more important. TOTO-1 dye will bind to almost any sequence in dsDNA.
TOTO binding with DNA is an equilibrium, where the dye can unbind if free DNA is added (i.e. equilibrium displacement) [4]. This is indeed the case when plasmids bearing TOTO enter in the cytoplasm where free DNA is present. Furthermore, TOTO can freely leak from the cytoplasm to the nucleus (where, again, free DNA is present) in a way similar to that reported for propidium iodide [4]. TOTO labelling DNA comprises a good tool for assaying the early steps of DNA entry into cells but cannot be used to investigate the process over a long period time. If this method is used to detect plasmid/membrane interaction (Figure 1), this approach is not adapted to study the intracellular traffic of plasmid. Indeed, in living or fixed cells, the fluorescent marker can dissociate from plasmid and label intracellular nucleic acids. This comprises the traffic of TOTO. Consequently, the visualization and quantification of plasmid biodistribution into cells can be biased. In our studies, to eliminate these artefacts, we used a dye covalently bound to the plasmid after checking that the level of protein expression was not affected. Indeed, we used the covalently rhodamine-labelled plasmid (pGeneGrip; Gene Therapy Systems, San Diego, CA, USA) to observe the cell traffic of fluorescent-labelled plasmid after electroporation [2].
Golzio et al. [2] demonstrated that this plasmid/membrane interaction is one of the key steps for obtaining gene expression. During the application Copyright 2009 John Wiley & Sons, Ltd.