Generation of Recombinant Viral Particles Using Embryonic Kidney Cells

Resumo

Source: Vijayraghavan, S., Kantor, B. A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells. J. Vis. Exp. (2017).

This video demonstrates the procedure for generating recombinant viral particles by transfecting embryonic kidney cells with plasmids encoding viral proteins and a gene of interest.

Protocolo

1. Transfecting Human Embryonic Kidney cells (HEK-293T) Using a Calcium Phosphate-based Protocol
   1. Transfection reagents
      1. To prepare 2x BES-buffered solution BBS (50 mM BES, 280 mM NaCl, 1.5 mM Na₂HPO₄), combine 16.36 g of NaCl, 10.65 g of BES (N, N-bis (2-hydroxyethyl)-2-amino-ethanesulfonic acid), and 0.21 g of Na₂HPO₄. Add double-distilled H₂O (dd-H₂O) up to 900 mL. Dissolve, titrate to pH 6.95 with 1M NaOH, and bring volume to 1 L. Filter via a 0.22 µM filter unit. Store at -20 °C.
      2. Prepare 1M CaCl₂. Filter solution via a 0.22 µM filter. Store at 4 °C.
2. Observe the plates that were seeded a day prior. Cells are ready for transfection once they reach 70 - 80% confluency.
3. Aspirate old media from the plates and gently add freshly-prepared media without serum.
4. Prepare the plasmid mix by aliquoting the four plasmids into a 15-mL conical tube. For a single 15-cm dish preparation, use 37.5 µg of the CRISPR/Cas9-transfer vector (pBK198 or pBK189), 25 µg of pBK43 (psPAX2-D64E), 12.5 µg pMD2.G, and 6.25 µg of pREV (Figure 1c).
5. Add 312.5 µL 1M CaCl₂ to the plasmid mix. To this, add up to 1.25 mL of sterile dd-H₂O.
6. Slowly (drop-wise) add 1.25 mL of 2x BBS solution while vortexing the mix. Incubate for 30 min at room temperature.
7. Add the transfection mixture dropwise to each 15-cm plate (2.5 mL per plate). Swirl the plates gently and incubate at 37 °C with 5% CO₂ for 2 - 3 h. Thereafter, add 2.5 mL (10%) serum per plate and continue incubating overnight (12 - 18 h).
   NOTE: Some labs use 3% CO₂ incubators to stabilize media pH. However, we do not observe any difference in transfection efficiency between 3% and 5% CO₂. Also, the size of CaPO₄ precipitates is crucial for transfection efficiency; the transfection mix has to be clear prior to its addition onto the cells. If the mix becomes cloudy during incubation, prepare fresh 2x BBS (pH = 6.95).

2. Day After Transfection

1. Observe the cells to ensure that they are nearing 100% confluency at this point with little to no cell death. Replace media by adding 25 mL of fresh DMEM + 10% serum to each plate. Continue incubating at 37 °C with 5% CO₂ for an additional 48 h.

Resultados

Figure 1: IDLV packaging. (a) Schematic of the wild-type integrase protein (b) The modified plasmid was derived from psPAX2. Representative agarose gel image of clones screened for mutated integrase clones...

Materiais

Lista de materiais utilizados neste artigo

Nome	Empresa	Número de catálogo	Comentários
100mm TC-Treated Culture Dish	Corning	430167
Tissue culture pipettes, 5 mL	Corning	4487
Tissue culture pipettes, 10 mL	Corning	4488
Tissue culture pipettes, 25 mL	Corning	4489
Hemacytometer with cover slips	Cole-Parmer	UX-79001-00
Human embryonic kidney 293T (HEK 293T) cells	ATCC	CRL-3216
293FT cells	Thermo Fisher Scientific	R70007
DMEM, high glucose media	Gibco	11965
Cosmic Calf Serum	Hyclone	SH30087.04
Antibiotic-antimycotic solution, 100X	Sigma Aldrich	A5955-100ML
Sodium pyruvate	Sigma Aldrich	S8636-100ML
Non-Essential Amino Acid (NEAA)	Hyclone	SH30087.04
RPMI 1640 media	Thermo Fisher Scientific	11875-085
GlutaMAX	Thermo Fisher Scientific	35050061
Trypsin-EDTA 0.05%	Gibco	25300054
BES (N, N-bis (2-hydroxyethyl)-2-amino-ethanesulfonic acid)	Sigma Aldrich	B9879 - BES
Gelatin	Sigma Aldrich	G1800-100G
psPAX2	Addgene	12260
pMD2.G	Addgene	12259
pRSV-Rev	Addgene	12253
lentiCRISPR v2	Addgene	52961
BES-buffered solution