Detection of DNA Replication Intermediates after Two-Dimensional Agarose Gel Electrophoresis Using a Fluorescein-Labeled Probe (original) (raw)

1999, Analytical Biochemistry

lipid-based reagent and electroporation, we conclude that two cell lines, J-774 and THP-1, are the easiest to transfect. Transfection efficiency is very low for HL-60 and U-937 cell lines. This difference in cell reactivity probably results from their respective physiological state. The physiological cellular state influences cellular response since once differentiated, THP-1, HL-60, and U-937 can no longer be transfected. This fact appears to be characteristic of differentiated myelomonocytic cells, as also observed by other authors (4). Therefore, if required, the introduction of exogeneous genes must be carried out prior to the differentiation process if their effects are studied in the differentiated state. This can be achieved by transfection for either transient or stable expression. We did not find that in the myelomonocytic cell lines tested the overall transfer efficiency of electroporation was much higher than that obtained by the tested lipid-based reagent. ␤-Gal-expressing cells did not exceed 3% of the viable cells which represented 20-30% of the initial cell number. On the other hand, with the amount of chemical reagent we used, no cytotoxicity was observed. If viable cell number is taken into account, the overall number of transfected cells is roughly equivalent in the two transfer techniques. High cell mortality caused by electroporation is also detrimental for the cell culture so that dead cells must be disgarded for further cell culture. A smaller number of cells are required for transfection with the chemical reagent that we tested (10 6 instead of 10 7 cells). This is particularly interesting when cells are difficult to grow. The amount of plasmid is also lower in gene transfer using lipid-based reagent (4 instead of 40 g). Finally, in actuality it is wise to use electroporation only for a stable expression of genes introduced in sensible myelomonocytic cells since this gene transfer technique may induce adverse responses and impair cellular functions. In summary, this communication reports a differential cellular response to transfection among the four myelomonocytic cell lines, J-774, THP-1, HL-60, and U-937, tested. We also conclude that transfection with a lipid-based reagent is more advantageous than electroporation because in addition to the absence of cytotoxicity, fewer cells and less plasmid are required with nevertheless equivalent transfer efficiency.