Invitrogen’s Optifect Transfection Reagent

Transfection techniques allow the introduction of macromolecules into eukaryotic cells, particularly nucleic acids. When DNA is introduced into the cell, a gene of interest can be studied by over-expression in the wild type or by insertion of a constituent mutant gene that encodes for either an active or inactive protein. Another popular method of molecular characterization is gene silencing induced by the introduction of double stranded RNA (dsRNA). The dsRNA is processed into short interfering RNA molecules that interrupt posttranscriptional gene expression via the degradation of sequence specific mRNA molecules. Transfection can be performed by chemical (i.e. calcium phosphate or cationic lipid delivery) or physical methods (introduction into the cell by electroporation, microinjection or high velocity projection). Of the options available, lipid cationic delivery provides an efficient and easy way to deliver nucleic acids into the cells.

The objective of lipid cationic delivery is to obtain the highest efficiency possible, while minimizing the level of cell death. Most of the reagents used require plating of the cells at a high confluence. This was an issue with the cells I studied, as the PC12 (rat phaeochromocytoma) cell line requires low confluence plating to achieve a phenotype of fully differentiated neurons when exposed to nerve growth factor and reduced amounts of serum. In addition, PC12 cells are hard to transfect and exhibit low efficiencies, when reported.

I tried several different reagents (Lipofectin, Lipofectamine, Lipofectamine 2000 and Optifect from Invitrogen,) and the PC12 cells were transfected with a bicistronic construct encoding for the gene of interest and for green fluorescent protein. The cells were plated at different confluences (0.5x10⁵ to 2x10⁵ cells per 35 mm Petri dish) and 18 hours later exposed to different ratios of plasmid and cationic lipid reagent (within the range suggested by the manufacturer).

Of the reagents tested, the highest transfection efficiency was recorded for Optifect. Lipofectamine 2000 had an 84% efficiency compared to that of Optifect, followed by lipofectamine with 45% and lipofectin with 36%. Also noted, Optifect was more suitable for transfections at the lower cell confluences needed for the PC12 cells to acquire a differentiated phenotype. I obtained my best results by plating 0.5x10⁵ cells per 35 mm Petri dish, 18 h prior to transfection. A 500 ul mixture (containing 4 ug of plasmid and 15 ul of Optifect) was prepared according to the manufacturer’s suggested protocol and then added to the cells in 1.5 ml of growing medium. After an overnight incubation, the cells received growth medium for 24 h and were then placed on differentiation medium. < The Optifect Transfection Reagent offered by Invitrogen is a very suitable and easy to use reagent for a hard to transfect cell line like PC12, especially when low confluence plating is required.

Stefan David, M.D.
Postdoctoral Fellow
Department of Neurology
University of Maryland at Baltimore