Polyethylenimine-Mediated Transfection of Embryonic Kidney Cells for Recombinant Virus Production

Begin with a solution of plasmids encoding the viral genome, the gene of interest, and viral proteins.

Add the plasmids to a solution containing polyethylenimine and mix well.

The positively charged polyethylenimine forms complexes with the negatively charged DNA.

Incubate to stabilize the complexes.

Add the complexes dropwise onto a monolayer of embryonic kidney cells.

Incubate to allow cellular uptake via endocytosis.

Polyethylenimine facilitates endosomal escape, releasing DNA into the cytoplasm.

Replace the medium to remove residual complexes and incubate.

Inside the cell, the plasmids direct the production of viral proteins and the viral RNA genome carrying the target gene.

Viral particles assemble in the cytoplasm and release into the medium.

Collect the supernatant containing viral particles and add fresh medium to sustain viral particle production.

Centrifuge the medium to remove debris, filter the virus-rich supernatant, and store it at a low temperature.

The production of lentiviral particles is a five day protocol that begins with splitting HEK293T cells to 60 to 80% confluency in a 162 square centimeter flask in cell culture medium. Incubate the cells overnight in a humidified cell culture incubator at 37 degrees Celsius. On the following day, prepare the DNA transfection solution and the polyethylenimine or PEI transfection solution as described in the protocol text.

Add the DNA transfection solution to the PEI solution. Vortex or invert a number of times, and incubate for five minutes at room temperature. Drip two milliliters of the DNA transfection plus PEI solution onto the HEK293T cells and incubate for four hours at 37 degrees Celsius.

After four hours, refresh the culture medium to remove the PEI. Incubate the cells for two days at 37 degrees Celsius. On day four, collect the supernatant in a 50 milliliter tube. Add new culture medium to the cells and return the flask to the incubator. Centrifuge the collected supernatant at 500 gs for five minutes to remove dead cells.

Then use a large 60-milliliter syringe to push the supernatant through a 0.45 micrometer filter. Store the filtered supernatant overnight at four degrees Celsius.