Delivering nucleic acids such as siRNA, mRNA, antisense agents, transcription factor decoys, and plasmid DNA offers a vast untapped potential for studying many new genetic factors for their relationship with various devastating human diseases. The barrier to the use of nucleic acids for the development of customized and specific treatments is safe, efficacious delivery of DNA.
Delivery method choice is an important factor that contributes to total transgene expression, level of throughput, and cell viability, which is especially critical in primary cells that are costly and difficult to obtain. Indeed, high cell viability becomes even more essential when transfection is carried out, in sensitive cell types, in vivo, or in ex vivo implanted cells.
Traditional means of carrying DNA into cells rely on cell membrane disruption such as electroporation or infection with viruses. Both of these have their own positive attributes and drawbacks but are generally not conducive to rapid high-throughput screening of multiple DNA constructs.
Other approaches relying on chemical agents such as lipids and polymers provide an improved means for high-throughput DNA screening in immortal cell lines, but few are designed with primary and sensitive cell types in mind.
Techulon has launched a new polymer-based transfection reagent, Glycofect™, which is designed specifically for DNA transfection in primary cells. Glycofect is a carbohydrate-containing polyamidoamine that binds with DNA of various sizes and forms positively charged nanoparticles called polyplexes. Glycofect has water-labile bonds that biodegrade during cellular uptake, enabling the release of DNA in a cell’s perinuclear region for maximum gene expression.
Studies have indicated that Glycofect is preferentially uptaken by cells via a caveolae-mediated endocytotic route, which contributes to the high nuclear delivery. Glycofect has been tested with various reporter plasmids and labeled oligo DNA and is proving to be an effective tool to the cell therapy and molecular biology communities.
In this article, we employ various means of testing gene expression and cell viability for Glycofect and several other commercially available transfection reagents in primary neonatal human dermal fibroblasts (HDFn), a cell of high therapeutic importance for its use in skin grafts and for making induced pluripotent stem cells.
We compare gene expression of various reagents based on transgene luciferase activity. Further, we quantify gene expression on a per cell basis with enhanced green florescent protein expression, using both florescent microscopy and flow cytometry to quantify results. Finally, to quantify the amount of healthy cells remaining after transfection, we test the viability of these cells using both the MTT and BCA assays.