Bio-Rad has developed a cell-line specific lipid transfection reagent, HEKFectin, that has been optimized for 293 cells (both adherent and suspension cultures). This includes the 293H, 293F and 293T cell lines. While HEKFectin works well over a broad range of cell densities, in the presence and absence of serum, the highest levels of reporter gene activity are achieved at densities from 70% to 90%. The recommendend three-step protocol requires no medium changes or serum-free incubation of the cells.
In our lab, we use HEKFectin for the production of lentiviruses (to infect 3T3-L1 cells). We employ a third-generation lentivirus vector system, derived from the human immunodeficiency virus (HIV-1), to generate particles for transformation of transgenes into 3T3-L1 fibroblasts and differentiated, non-dividing 3T3-L1 adipocytes. This lentivirus system uses only a fractional set of HIV-1 genes (gag/pol for capsid proteins and rev for reverse transcriptase) and the lentiviruses are pseudotyped with the glycoprotein from the vesicular stomatitis virus (VSV-G) for infection. The different genes are encoded on three separate vectors and another plasmid carries the lentiviral expression cassette, containing the gene of interest. Production of a lentivirus involves the simultaneous transfection of HEK293T host cells with all four vector constructs using HEKFectin and harvesting of infectious viral particles from the culture supernatant.
The procedure for transfection with HEKFectin is very simple. The cells were plated to a density that would yield around 70-80% confluency the following day. The appropriate amount of HEKFectin was added to serum-free medium in a polystyrene container and the DNA was separately diluted in serum-free medium to the final concentration. The DNA and the HEKFectin solution were mixed together and incubated for 20 min at room temperature to establish complex formation. The mixture was added directly to the cells in serum-containing medium. In our hands 16 ul HEKFectin with approximately 5 ug of DNA (total) was sufficient for transfection of adherent cells in a 25 cm2 dish. The supernatant was harvested 48h and 72h post-transfection, passed through a 0.45 um filter and the viral concentration was measured by an antigen-capture ELISA for HIV-1 p24 protein. For infection of 3T3-L1 cells we exposed the fibroblasts to MOI=1 and the adipocytes to MOI=3 in the presence of Polybrene (Sigma) and incubated over night. The efficiency was analyzed 3-6 days post-transduction by confocal fluorescence microscopy for GFP expression.
We found that viral production using HEKFectin was a rapid, efficient and highly reliable method compared to the classic calcium phosphate procedure. We transfected the HEK293T cells using the supplied reagents from the kit. A lentiviral backbone was also added where the gene for green fluorescence protein (GFP) is cloned downstream of the human phosphoglycerate kinase (PGK) promoter. After 24h post-transfection, we detected GFP-positive cells and the intensity increased to a maximum after 48h. As judged by GFP staining, the transfection efficiency of HEK293T cells was approximately 80%. We observed no cytotoxicity and the cells were growing similar to the non-transfected controls. The virus titer reached approximately 300 ng/ml, corresponding to 7.5 x 105 infectious particles per ml. GFP-lentiviruses were then used to infect 3T3-L1 fibroblasts and differentiated 3T3-L1 adipocytes and after 5 days in culture the cells were analyzed by fluorescence microscopy. Nearly all cells were GFP positive.
HEKFectin permits highly efficient transfection of four different plasmids in one reaction, resulting in a consistently increased yield in intact, biologically active lentivirus particles compared to the conventional calcium phosphate transfection. The application of HEKFection is rapid because there is no serum-free incubation step, resulting in a time savings and an increase in viral production.
Ulrike Schmidt
PhD student
German Institute of Human Nutrition, Rehbrücke