Targefect-RAW is a new reagent from Targeting Systems specifically designed for the transfection of the RAW 264.7 macrophage-like cell line. Although these cells are quite useful for studying innate immune responses and numerous other processes, they are extremely difficult to transfect by calcium phosphate, electroporation, and lipid-based delivery methods. Thus, reproducible transient transfection of reporter plasmids and cDNAs proves difficult, if not nearly impossible, and the creation of stable RAW transfectants is limited. To address these limitations, Targefect-RAW combines lipid-based transfection with a novel component, Virofect, to significantly enhance delivery and subsequent expression of plasmid DNA.
Virofect (also available from Targeting Systems) is a replication deficient, adenovirus-derived enhancer formulation that greatly augments transfection efficiency in a variety of cell types when used in combination with additional Targefect reagents, as well as other cationic reagents such as Lipofectamine (from Invitrogen) and Superfect (from Qiagen). Virofect enhances gene expression by utilizing adenoviral receptors on the cell surface to increase uptake and intracellular delivery of transfection complexes, while also preventing lysosomal degradation of the complexes. When used in combination with Targefect-RAW reagent, Virofect dramatically enhances plasmid delivery and expression, providing an easy to use, highly superior tool for RAW cell transfection.
For transfection, cells are grown to 70% confluency in 12-well plates. Complexes are then prepared by combining 6 ug DNA with 12 ul Targefect-RAW and 24 ul Virofect in 600 ul of serum-free DMEM. After incubation for 20 minutes at 37„aC, 125 ul of complexes are then diluted in an equal amount of complete medium and added to the cells for 2 hours at 37„aC. 600-800 ul complete medium is then added to cells, and they are further incubated for 24 ¡V 36 hours before assay. Targefect-RAW transfections can be easily scaled for any size plate or dish and are comparable in time and difficulty to other commercial transfection protocols.
We have successfully used Targefect-RAW to overexpress cDNAs and reporter plasmids in RAW cells, with transfection efficiencies usually within the 60 ¡V 80 % range. Most other commercial reagents transfect around 0.1 ¡V 1 % of cells in our hands, so the effectiveness of Targefect-RAW is clear. Even compared head-to-head with Lipofectamine LTX, which performs much better than Lipofectamine 2000 or Fugene 6, Targefect-RAW is substantially more effective (see fluorescence activated cell sorting and IF data below). In addition, Targefect-RAW out-performs Amaxa Nucleofector technology in transient transfections, with significantly decreased toxicity and user cost.
Overall, Targefect-RAW is an affordable and highly efficient RAW cell transfection reagent that provides reproducible transfections on the order of those seen in fibroblast cell lines. Since Virofect contains replication-deficient adenovirus, users must practice care when working with the reagent; however, this inconvenience is heavily outweighed by the stellar performance of the product.