Lentiviruses can be used in research applications to transduce a wide range of cell types and stably integrate genetic payload into the host genome, allowing for long-term studies in vivo. This protocol makes use of FuGENE® HD Transfection Reagent and newly available components to eliminate toxicity associated with lentiviral preparations and consistently produce titers in the mid to high 1010 transduction units (TU) per milliliter.
Lentiviruses can be used in research applications to transduce a wide range of cell types and stably integrate genetic payload into the host genome, allowing for long-term studies in vivo. This protocol makes use of FuGENE® HD Transfection Reagent and newly available components to eliminate toxicity associated with lentiviral preparations and consistently produce titers in the mid to high 1010 transduction units (TU) per milliliter.Materials
This protocol is for one Nalge Nunc Nunclon T175 flask. Typically, 5 to 10 flasks are required for applications that require the highest titer. Scale reagents depending on desired size of preparation.
Packaging Cell Line: HEK 293-FT (Invitrogen). This adherent cell line is amenable to serum-free media formulations without diminished FuGENE® HD Reagent transfection efficiency. The use of serum during virus production is not compatible with achieving virus preparations free from cytotoxicity. HEK-293FT cells should be switched from the manufacturer's recommended medium (DMEM) to Opti-MEM (Invitrogen) with GlutaMAX supplemented with 5% fetal bovine serum (FBS) and 500 µg/ml G418 for routine growth and maintenance. Maintain culture vessels at 37° C at 5% CO2 in 95% RH.
Expression Vector: cFUGW (Cal Tech), or other vectors compatible with third generation lentivirus packaging system (most commercially available expression vectors). Vectors should encode eGFP or the appropriate transgene, WPRE, and cPPT; the cPPT and WPRE elements considerably increase transgene expression and titer, especially in primary cells.
Packaging Plasmids: pLP1, pLP2, and pLP/VSV-G (Invitrogen). These modified packaging plasmids allow expression in trans of proteins required to produce functional virus, are resistant to recombination, and contain optimized promoter and enhancer elements for expression in HEK-293FT cells.Methods Lentivirus Supernatant Production Day 1
1. Coat T-175 flask(s) with 10 ml polylysine solution (0.01 mg/ml, or 10 µg/ml) in sterile water/PBS.
Leave for at least 1 h.
2. Wash twice with 20 ml sterile water.
3. Split HEK-293FT cells into coated flasks at ∼50% confluency around 5:00 PM the day before transfection, with cells growing in 20 ml of 10% FBS Opti-MEM (with GlutaMAX).Day 2
4. Next morning (9:00 AM), remove media, add 20 ml serum-free (SF) Opti-MEM (with GlutaMAX) supplemented with 25 µM chloroquine.
5. Prepare transfection complex:
Add together 10.5 µg pLP1, 7 µg pLP2, 10.5 µg pVSV-G, and 9.3 µg cFUGW. Plasmid preparations should be of extremely high quality and endotoxin free.
Dilute DNA into 2 ml of SF Opti-MEM.
Add 100 µl FuGENE® HD Transfection Reagent, briefly vortex, and incubate 15 minutes at room temperature.
6. Pipet directly to media in the flask of cells. Agitate flask until contents are distributed.
7. In the early evening of the same day (∼5:00 PM), supplement with 10 µM sodium butyrate, directly to the flask medium.Day 3
8. Next morning (9:00 AM), discard media and replace with 20 ml of SF Opti-MEM (with GlutaMAX and no other supplements). Note: the discarded media has a small amount of virus and is BSL-2 (bleach disposal, etc.).Day 4
9. Next morning, transfer the media in each flask (∼20 ml) to a Falcon 50 ml centrifuge tube. Replace 20 ml of fresh SF Opti-MEM (with GlutaMAX) media to flask.
10. Spin Falcon tube at 2000× g for 10 min at 4° C and transfer supernatant to new 50 ml tube. Store at 4° C overnight.Day 5
11. Next morning (9:00 AM), collect ∼20 ml of media to a new 50 ml tube.
12. Spin at 2000×g for 10 min at 4°C and then transfer supernatant to the existing 50 ml tube from the day before, which will now contain approximately 40 ml of viral supernatant.
13. Pass tube contents through a 22 µm vacuum filter (Millipore).
14. Virus can be used as a 1× virus preparation (∼1× 107 TU/ml); freeze as 5 ml aliquots at -80° C.
To determine whether this protocol produced sufficiently pure and concentrated virus for in vivo applications, a 2-µl volume of 4 × 1010 TU virus encoding eGFP was injected into the anterior region of the mouse caudate putamen. After 1 week, brain sections were analyzed through immunohistochemistry and immunofluorescence. Broad infectivity with minimal toxicity around the needle site and through the striatum demonstrates the high quality of lentiviral preparations produced using FuGENE® HD Transfection Reagent.
For more detailed information on optimizing lentivirus production (including steps for titer concentration) from the authors of this protocol, see Tomlinson, R.L. and West, A.B. 2008. Optimized Production of Lentivirus Using FuGENE® HD Transfection Reagent. Biochemica 3/2008: 17-19, at www.roche-applied-science.com.
FuGENE is a registered trademark of Fugent, L.C.C., USA. Other brands or product names are trademarks of their respective holders.Correspondence
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