If you are new to lentivirus, please read this very helpful guide article on lentivirus. For safety reasons, lentiviral vectors used for research purpose has been engineered to be replication incompetent and often also self-inactivating after integration. Please use biosafety procedures BSL2 or higher for lentivirus work.

Reagents

For tissue culture:

• 293 cells (e.g., Takara, Cat# 632273)
• DMEM (e.g., Thermo Fisher, 11965118)
• Fetal bovine serum (FBS; e.g., GE Healthcare/Cytiva, SH30070.03)
• 200 mM L-glutamine (e.g., Thermo Fisher, 25030081)
• 100x PenStrep (10,000 units/mL penicillin, 10,000 µg/mL streptomycin; e.g., Thermo Fisher, 15140163)
• DMEM/F-12 (e.g., Thermo Fisher, 11039047)
• 25 mM chloroquine in PBS (e.g., MilliporeSigma, C6628)

1. To make DMEM Complete medium for 293 cells, add 50 mL FBS, 5 mL L-Glutamine and 5 mL 100x PenStrep to 500 mL DMEM. Filter sterilize and store at 4°C.

2. To make ~20 mL 25 mM chloroquine in PBS, weigh ~250 mg chloroquine diphosphate salt in a 50 mL tube. Assuming X mg is weighed, the volume (mL) of PBS to add: V = X / 12.8965. Fully dissolve by vortexing, filter sterilize, divide into 100 µL or 1 mL aliquots and store at -20°C.

Plasmids for transfection:

• Packaging plasmid psPAX2
• VSV-G envelope plasmid pMD2.G
• Your lentiviral transfer plasmids (e.g., sgRNA-expressing plasmids made from pW212)

Reagents for transfection, concentrating, and titer estimation:

• 1 M HEPES-NaOH, pH 7.50
• 5 M NaCl
• 150 mM Na₂HPO₄
• 2 M CaCl₂
• 0.45 µm Steriflip filter (MilliporeSigma, SE1M003M00)
• 5x PEG-it reagent (System Biosciences, LV825A-1)
• Lenti-X GoStix Plus (Takara, 631281)

1. To make 0.3 M CaCl₂, mix 30 mL 2 M CaCl₂ with 170 mL H₂O (Millipore purified water or Ultrapure water). Filter sterilize and keep at 4°C.

2. To make 2x HBS pH 7.10:

Working concentration	Stock concentration	Volume for 200 mL
H2O	-	*176.8 mL
50 mM HEPES	1 M	10 mL
280 mM NaCl	5 M	11.2 mL
**1.5 mM Na2HPO4	150 mM	2 mL

*use 150 mL first; adjust pH to 7.10 with NaOH; then adjust volume to 200 mL **Na₂HPO₄-7H₂O, MW = 268.07 g/mol

After adjusting pH and volume, filter sterilize and keep at 4°C. Periodically check the pH of 2X HBS using pH paper, since correct pH is the key for efficient transfections.

Procedure for lentivirus packaging

1. Day 1: Seed 293 cells for transfection

• For each virus to make, seed 5 million 293 cells per 10-cm dish, or 12.5 million 293 cells per 15-cm dish.

  • The number of cells to seed may need to be adjusted. I used to be using 4 million cells per 10-cm dish, but I had to increase the number after using a new cell counter, which seems to segment the cells better and count the cell number more accurately.
  • Packaging with a 10-cm dish is often sufficient, but a 15-cm dish may be preferred for lentiviral vectors expected to be more difficult. For example, if the lentiviral vector is large so that the between LTR length is approaching the packaging limit.
  • 293 cells are used for lentivirus packaging because they are very easy to transfect. We routinely get >80% transfection efficiency with classical calcium co-precipitation method using 293TN from System Biosciences or AAV-293 from Takara Clontech (Takara, Cat# 632273).

2. Day 2: Transfection

• Check seeded cells: they should be ~80% confluent.
  • If cells are slightly low confluency, wait for half a day before transfecting. Cells can be transfected within 50-80% confluence, but the more cells you have the higher titer you will get.
  • If the cells are apparently slow growing, consider thawing early passage stocks.
• If cells are ready:
  • Warm up 2x HBS (pH = 7.10) and 0.3 M CaCl2 solution to room temperature.
  • Thaw the stock plasmid DNAs: your lentiviral vector, the packaging plasmid psPAX2 and the envelope plasmid pMD2.G.
  • Warm up enough DMEM Complete medium at 37°C. Each 10-cm dish will cost 24 mL medium in total, whereas each 15-cm dish will cost 60 mL medium in total.
  • Thaw an aliquot of 25 mM chloroquine (1000x) that is sufficient for the medium.
• Transfection for 10-cm dish:
  • In a tissue culture hood, add chloroquine to DMEM Complete medium for a final 25 µM concentration. Replace medium in each dish with 8 mL fresh medium with 25 µM chloroquine. Put the cells back in the incubator.
  • For each dish to transfect, prepare two 15 mL tubes: (1) remove their lids; (2) label one tube with the transfer plasmid to transfect.
  • In the unlabeled set of 15 mL tubes, add 1 mL 2x HBS to each tube.
  • In the labeled set of 15 mL tubes, add 10 µg each of the lentiviral transfer plasmid, psPAX2 and pMD2.G.
  • Take up to 4 dishes each time to the hood. Process them one by one for transfection.
  • For each transfection, use a 5 mL pipet to add 1 mL 0.3 M CaCl₂ to the 15 mL tube containing the plasmid DNA mixture. Pipet to mix well. Add this mixture into the tube containing 2× HBS drop by drop. Pipet to mix well.
  • Immediately add this mixture drop by drop into a 10-cm dish for transfection.
  • After 6-12 hours of transfections (can be overnight), replace medium with 8 mL fresh medium + 25 µM chloroquine.
• Transfection for 15-cm dish:
  • In a tissue culture hood, add chloroquine to DMEM Complete medium for a final 25 µM concentration. Replace medium in each dish with 20 mL fresh medium with 25 µM chloroquine. Put the cells back in the incubator.
  • For each dish to transfect, prepare two 15 mL tubes: (1) remove their lids; (2) label one tube with the transfer plasmid to transfect.
  • In the unlabeled set of 15 mL tubes, add 2.25 mL 2x HBS to each tube.
  • In the labeled set of 15 mL tubes, add 22.5 µg each of the lentiviral transfer plasmid, psPAX2 and pMD2.G.
  • Take up to 4 dishes each time to the hood. Process them one by one for transfection.
  • For each transfection, use a 5 mL pipet to add 2.25 mL 0.3 M CaCl₂ to the 15 mL tube containing the plasmid DNA mixture. Pipet to mix well. Add this mixture into the tube containing 2× HBS drop by drop. Pipet to mix well.
  • Immediately add this mixture drop by drop into a 15-cm dish for transfection.
  • After 6-12 hours of transfections (can be overnight), replace medium with 8 mL fresh medium + 25 µM chloroquine.

Day 3-4: Collect virus-containing supernatant

• Warm up medium with 25 µM chloroquine prepared on the day of transfection.
• At ~24 hour post the first medium change, collect the supernatant into a 50 mL tube, and add back 8 mL fresh medium + 25 µM chloroquine (or 20 mL for 15-cm dish). Store at 4°C.
• After another 24 hours, collect the supernatant into the same 50 mL tube stored at 4°C.
  • When testing new constructs, the Lenti-X GoStix Plus (Takara, 631281) can be used as a handy tool to estimate the viral titer at this point. The Lenti-X GoStix Plus can also be used to examine the titer after the virus is concentrated.
• Filter the pooled supernatant through a 0.45 µm SteriFlip filter.
  • If not concentrating the virus, the filtered solution can be directly used, or stored at -80°C after freezing in liquid N₂ or dry ice.
• To concentrate virus, add ~4 mL 5x PEG-it reagent (or ~10 mL for 15-cm dish format), mix well by pipetting or inverting. Refrigerate for at least 12 hours at 4°C. Virus solution with PEG is stable for up to 5 days at 4°C.

Day 5: Collect concentrated virus

• Check the virus + PEG solution. With PEG and refrigerating, it should become turbid as virus precipitates from the solution.
• Pre-cool a swinging-bucket centrifuge to 4°C.
• Spin virus + PEG solution at 1500x g for 30 min at 4°C to pellet the virus.
  • There should be a visible light yellow to white colored pellet.
  • Keep the tubes on ice during transfer.
• In a tissue culture hood, remove the supernatant as much as possible by aspirating while tilting the 50 mL tube. Be careful not to touch the pellet.
• Resuspend the pellet with 1/10 to 1/100 of the original viral solution volume of DMEM/F12 medium (OK with PenStrep). I typically use 500 µL.
  • The Lenti-X GoStix Plus (Takara, 631281) can be used to estimate the viral titer at this point after 1:100 dilution of the viral stock.
  • Empirically, a GoStix value of 50 is equivalent to ~5 x 10⁵ IFU/mL.
  • The typical yield of a 10-cm dish packaging of lenti-sgRNA using pW212 backbone is 500 µL at 1.5 x 10⁸ IFU/mL.
• Store at -80°C.
  • Aliquoting is not necessary, as PEG provides cryoprotection for the solution, and data has shown freeze-thaw for up to 5 times do not seem to affect the virus titer.