Ingenious Transfection Reagents: Getting Your Message Across

All types of transfection reagents share a variety of challenges, however. Non-targeted genes can be accidentally up- or down-regulated by the insertion of your exogenous genetic material, potentially complicating the interpretation of your results. Cells in primary culture are notoriously difficult to transfect, regardless of the reagents used. The heterogeneity in transfection efficiency that different cell types pose can also be a challenge for labs who work with multiple cell types. "It is impossible to find one reagent that achieves superior transfection for all cell types," says Kathy Evensen, Product Development and Technical Support Specialist Mirus Bio Corporation. "Another obstacle is choosing a reagent that achieves superior transfection efficiency but does not cause cellular toxicity," Evensen adds.

Another lipid-based reagent is FuGENE 6 manufactured by Roche Applied Science, which claims that their reagent can help you circumvent some optimization steps when transfecting different cell lines. "FuGENE 6 Transfection Reagent has been shown to provide high transfection efficiency in more than 700 cell types including many primary cell lines when used at a ratio of 2-3," says Roche Applied Science’s Product Manager Jeffrey Emch. Roche also claims that in addition to unusually low cytotoxicity, their FuGENE 6 minimizes the nonspecific gene alteration that is a hazard of this method. "A recent gene expression study demonstrated that FuGENE 6 Reagent significantly influenced the expression of only 30 non-target genes," comments Emch, "compared with the more than 2000 non-target genes affected by a leading competitor’s transfection reagent."

In the fall of 2005, look for Roche’s FuGENE HD Transfection Reagent. This new product builds on the strengths of FuGENE 6 and is designed for application to cell lines that have traditionally been considered difficult to transfect. Currently Roche offers their X-tremeGENE Q2 transfection reagent for the difficult-to-transfect K-562 and Jurkat cell lines.

A similar compaction of the genetic material is achieved by Qiagen’s SuperFect Reagent, comprised of activated dendrimer molecules that radiate outward spherically from a central core. Positively-charged amino groups at the ends of the radiating dendritic branches interact with the negatively-charged phosphate groups of the nucleic acids. The SuperFect Reagent molecules pack DNA into compact structures that bind to the cell membrane and are taken up by endocytosis. Qiagen claims better reproducibility in transfection results compared to its competitors due to the precisely controlled shape and size of the engineered dendrimer molecules.

Also relying on endocytosis for gene delivery, QBiogene boasts 100% transfection rates for tumor cell lines (Neuro 2A, Jurkat, and K562) using its DuoFect transfection system, which takes advantage of the endocytosis of ubiquitous transferrin receptors. Human transferrin, covalently linked to PEI and its tightly compacted DNA, is taken up by transferrin receptor-mediated endocytosis. Once inside the endosomes, PEI upsets the pH balance such that the endosome lyses, releasing the DNA into the cytoplasm. DuoFect efficiently transfects other cell types as well, such as HeLa, CHO, and COS cells.

OZ Biosciences offers a magnetic twist using a technology they call Magnetofection – using magnetism to augment transfection efficiency. Your genetic material of interest is incubated with magnetic nanoparticles coated with cationic molecules. The complexes that form between these species are then transfected into cells in the presence of a magnetic field. OZ Biosciences claims that with Magnetofection, the complexes containing the genetic material become concentrated on the cells within a few minutes, such that 100% of cells receive a dose of genetic material. OZ Biosciences sells four types of Magnetofection-based reagents. Their PolyMag reagent can be used with any type of nucleic acid, while SilenceMag (see below) is aimed at siRNA delivery. CombiMag is designed to be used with other transfection reagents, and ViroMag is geared toward use with viral infections. According to OZ Biosciences, Magnetofection can make transfection more effective while also decreasing toxicity. “Magnetofection is the perfect solution for transfection of primary cells and hard-to-transfect cell types,” says Laurent Meunier, a scientist at OZ Biosciences. “It is the only technology applicable to all nucleic acids and all non-viral and viral vectors.”

Roche Applied Science makes the X-tremeGENE siRNA Transfection Reagent for the transfection of common cells lines, as well as difficult-to-transfect cell types such as HT29 human adenocarcinoma cells. Roche claims over 90% knockdown efficiency in many cell types. With another magnetic twist on siRNA delivery, OZ Biosciences’ SilenceMag is a Magnetofection-based reagent specifically designed for siRNA experiments. Importantly, it performs well even at low doses of siRNA (<10 nM), and is effective at silencing expression in cell lines, primary cells, and other difficult-to-transfect cell types. And Mirus Bio offers two reagents for siRNA transfection – their tried and true TransIT-TKO Transfection Reagent, and their newer TransIT-siQUEST Transfection Reagent, specially designed for siRNA transfection. Mirus Bio maintains that together, the two reagents give researchers more flexibility depending on the cell type used.

In vivo transfection has already made its way from the bench to the clinic. Edensen says that “delivery of a cytotoxic gene (diphtheria toxin) behind a tumor specific promoter (H-19) using in vivo-jetPEI to treat human bladder cancer has shown extensive tumor regression for treated patients.” Two more human clinical trials are on the horizon as well – one combines apoptosis and chemotherapy to treat pancreatic cancer, and the other is geared toward developing a method for genetic vaccination by topical gene delivery to antigen-presenting Langerhans cells of the skin. The latter study shows promise for the treatment of HIV infection by DNA immunization. It is hoped that such experiments using in vivo transfection can pave the way toward gene therapies for a multitude of diseases.