The DOTAP Liposomal Transfection Reagent from Roche Applied Science is a liposome formulation of the monocationic lipid DOTAP that can be used for the highly efficient transfection of DNA into eukaryotic cells for transient or stable gene expression. DOTAP Liposomal Transfection Reagent is also suitable for the efficient transfer of other negatively charged molecules, such as RNA, oligonucleotides, nucleotides, ribonucleoprotein (RNP) complexes, and proteins, into mammalian cells.
We have been using DOTAP in our laboratory for almost a year now. The use of DOTAP was shown to be instrumental in the induction of a strong and specific CD8+ CTL response against different cancer tumor cells. While the intrinsic toxicity of DOTAP for DCs was low, its function was presumably to facilitate the cytoplasmic incorporation of exogenous antigen for major histocompatibility complex class I - restricted presentation to CD8+ T cells. To support this notion, we found it more difficult to induce specific CTLs against autologous tumor targets when we used DC pulsed overnight with recombinant protein without DOTAP.
A number of methods have been developed to transfer DNA into eukaryotic cells for the study of gene regulation and gene expression. Among them are the use of calcium phosphate or other divalent cations, polycations, liposomes, retroviruses, microinjection and electroporation. However, all of these methods suffer from one or more problems related to cellular cytotoxicity, poor reproducibility, inconvenience or insufficiency of DNA delivery. Mixing the DOTAP Liposomal Transfection Reagent with DNA results in a spontaneously formed stable complex that can be directly added to the tissue culture medium with or without serum. These complexes adhere to the cell surface, fuse with the cell membrane, and then release the DNA into the cytoplasm. It can also be successfully used for in vivo applications. As previously mentioned, this method of DNA transfer is very gentle, avoiding cytotoxic effects, so that the cells are transfected with high efficiency. However, from our own experience, how well DOTAP works depends on the system. In particular the cell type (e.g. dendritic cells vs. lymphoblastoid cell lines) must be experimentally tested and careful optimization and standardization of the transfection conditions for each cell type is required. Cells used for transfection experiments should be healthy, well proliferating and seeded at a constant density to minimize both the intra- and interassay variability.
Working instructions are easy to follow. Usually, cells to be transfected are incubated with the DOTAP/nucleic acid mixture for 3-10 hours. Depending on the cell type used, cells may be cultured in culture medium containing the DOTAP/nucleic acid mixture for up to 72 hours with no appearance of cytotoxic side effects. We always monitored the transfection efficiency by determining the expression level of a reporter protein by different experimental parameters depending on the reporter system used.
We believe that Roche’s DOTAP Liposomal Transfection Reagent is a highly effective tool for the trasfection of DNA, RNA, oligonucleotides, ribonucleoprotein particles and proteins that could be routinely used by researchers working on transfection protocols. It is definitely an asset for the laboratory specialized on the transfection of eukaryotic cells for their experiments.
Alisher R. Dadabayev, MD
Postdoctoral Research Associate
Laboratory of Immunology
Department of Internal Medicine
TTUHSC, Amarillo