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

Resumo

Source: Van Lidth de Jeude, et al. A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids. J. Vis. Exp. (2015).

This video demonstrates the process of producing viral particles through transfection of embryonic kidney cells with plasmid–polyethylenimine complexes, which leads to viral genome expression, particle assembly, and virus collection in the medium.

Protocolo

1. Preparation of Polyethylenimine (PEI) as Transfection Reagent

1. Dissolve approximately 150 mg of PEI into 100 ml of H₂O.
2. Adjust the solution to pH 7.4 by adding HCl until it becomes clear and stirring until completely dissolved. This may take between 10 and 60 min. Add water to an end concentration of 1 mg/ml.
3. When clear, filter the PEI solution through a sterile 0.22 µm filter and store in aliquots of 5 ml in a -80 °C freezer.

2. Production of Lentiviral Particles

Day 1:

1. Split HEK293T cells to 60%-80% confluency in 162 cm² flask or large petri dish in cell line culture medium (DMEM supplemented with 10% FCS(Fetal calf serum), 1% penicillin/streptomycin and 2 mM glutamine supplement).

Day 2:

2. Prepare a DNA transfection solution containing 45 µg of total plasmid DNA(Deoxyribonucleic acid) by adding together lentiviral packaging vectors (7 µg of pVSVg; 5 µg of pRSV rev; 13 µg of pMDL) and 20 µg of lentiviral plasmid encoding the gene of interest or shRNA(Short hairpin Ribonucleic acid)of interest.
3. Adjust to a volume of 1 ml using DMEM(Dulbecco's modified eagle medium).
4. Prepare PEI transfection solution by adding 90 µl of 1 mg/ml PEI to 930 µl of DMEM and incubating for 5 min at room temperature.
5. Add DNA transfection solution to PEI solution. Vortex or invert a number of times and incubate for 5 min at room temperature to obtain DNA transfection solution.
6. Drip 2 ml of the DNA transfection solution onto the HEK293T cells and incubate for 4 hr in a humidified cell culture incubator on 37 °C.
7. After 4 hr, refresh the culture medium to remove PEI. It is not necessary to wash cells before adding a new medium.
   NOTE: PEI is cytotoxic and incubation times longer than 4 hr may cause harm to the HEK293T cells.

Day 4:

7. Replace supernatant with new culture medium. Keep supernatant (containing virus); this will be used in step 2.10.
8. Put the supernatant in a 15 ml flask. To remove dead cells, centrifuge for 5 min at 500 x g.
9. Push supernatant through 0.45 µm filter using a large 60 ml syringe. Store overnight at 4 °C.

Day 5:

10. Collect the second batch of supernatant; centrifuge and filter as in steps 2.8-2.9.
11. Pool the supernatants from steps 2.9 and 2.10 in ultracentrifuge tubes and centrifuge at 50,000 x g in an ultracentrifuge for 90 min.

Materiais

Lista de materiais utilizados neste artigo

Nome	Empresa	Número de catálogo	Comentários
Polyethyleneimine	Polysciences	23966-2
DMEM medium	Lonza	BE12-614F
Fetal calf serum	Lonza	DE14-801F
Penicillin-streptomycin	Invitrogen	15140-122
Glutamin	Invitrogen	25030-024
Matrigel	BD	BD 356231
Advanced DMEM-F12	Gibco	12634-010
N2	Invitrogen	17502-048
B27	Invitrogen	17504-044
N-acetyl cysteine	Sigma	A9165-1G
Mouse Egf	Invitrogen	PMG8045
Hepes 1 M	Invitrogen	15630-056
Glutamax 100x	Invitrogen	35050-038
Chir 99021	Axon	1386
Y27632	Sigma	Y0503-5MG
Polybrene	Sigma	107689
Nicotinamide	Sigma	N0636
Trypsin	Lonza	BE02-007E
Puromycin	Sigma	P 7255