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Package of Lenti-shRNA virus using HEK293T cells

1. Before seeding cells for virus package, make sure HEK293T cells are in rapid replication state by growth cells to approximately 75%~80% confluence in a 10 cm cell culture dish and passaging at 1:2 ratio for at least 2 consecutive days
2. On day 0, Seed HEK293T cells at 8.5~9?106 cellsin growth medium without antibioticsfor a 10-cm culture dish to reach around 90% confluence on next day
3. On day 1, Co-transfect pGIPZ-shRNA or pLOC-ORF with pCMV-DR8.2 and pCMV-VSV-G (ratio:1:1:0.1 or 0.2) into HEK293T cells (adjust medium with 5 mL)
   1. Mix the plasmid DNAs (as indicated below) in 0.5 mL Opti-MEM 
      1. pCMV-D8.2: 10mg
      2. Lenti-shRNA: 10 mg
      3. pCMV-VSVG: 1 mg
   2. Dilute Lipofectamine 2000 with Opti-MEM: 50 ml of Lipofectamine in 0.5 mL Opti-MEM
   3. Incubate at room temperature for 5 min, thencombine the DNA mixture with diluted Lipofectamine 2000
   4. After 20 min incubation, Add the DNA-Lipofectamine mix to the cells (1 mL to 5 mL, the total medium will be ~6 mL)
4. Change medium within 18 hours (remove the old medium, add 10 mL fresh growth medium)
5. Check GFP expression around 24 hours after transfections. If GPF+ cells <50%, transfections are failed and please try again. Best practice is GFP+ cells>80-90%.
6. Harvest medium 48 hours after the medium change
7. Optional: harvest medium again 24 hours after harvesting the first batch of medium
8. Perform virus concentration using Lenti-X Concentrator by following the protocol from Clonetech or home-made virus concentrator
9. Aliquot and Store the concentrated lentivirus at -80 degrees. Avoid multi-cycle of freeze and thaw. One cycle will lead to 50-90% loss of lentivirus.

Protocol for different transfection reagents for lentivirus package 

Lipofectamine (for 10cm dish)

• Change medium with fresh OPTI-MEM (5mL/10cm dish)
• Dilute DNA plasmids into 500?l OPTI-MEM and Lipofectamine2000 in 500?l, respectively
• Incubate at room temperature for 5 min
• Add diluted lipofectamine to DNA mixture
• Incubate at room temperature for 20 min
• Add the lipofectamine-DNA mixture onto cells drop-wise
• Replace transfection medium with fresh completely culture medium after 5 hours
• Change medium next day (about 18 hours culture)
• Collect the supernatant on 24 hours and on 48 hours after changing medium

Xfect Polymer (for 10cm dish)

• Change medium with fresh culture medium containing FBS but no antibiotics (5mL/10cm dish)
• Dilute DNA plasmids into 600?l Xfect Reaction Buffer and vortex for 5 sec at high speed followed by brief spinning down
• Add 6.3 ?l Xfect Polymer to the diluted DNA mixture directly
• Incubate at room temperature for 10 min
• Add the Xfect-DNA mixture onto cells drop-wise
• Replace transfection medium with fresh completely culture medium after 5 hours
• Change medium next day (about 18 hours culture)
• Collect the supernatant on 24 hours and on 48 hours after changing medium

Note: Thaw the reagents before use and then store at 4 ¡ãC.

LipoD293 (for 10cm dish)

• Change medium with fresh complete medium containing FBS & antibiotics (5mL/10cm dish) 0.5~1.0 hour transfection
• Dilute DNA plasmids into 500?l Serum-free DMEM with high glucose (i.e. Hyclone DMEM), vortex gently and briefly spin down to bring drops to the bottom of the tube
• Dilute 46 ?l LipoD293 transfection reagent with 500 ?l serum-free DMEM (Hyclone DMEM), vortex gently and spin down briefly
• Add the diluted LipoD293 Reagent immediately to the diluted DNA mixture, mix well by pipette up and down 3~4 times
• Incubate at room temperature for 15 min
• Add the 1000 ?l LipoD293-DNA complex drop-wise onto the cells
• Replace transfection medium with fresh completely culture medium after 5 hours
• Change medium next day (about 18 hours culture)
• Collect the supernatant on 24 hours and on 48 hours after changing medium

Note

1. Never use Opti-MEM to dilute DNA and LipoD293 reagent because it will disrupt transfection complex;
2. Add dilute LipoD293 to diluted DNA mixture not in a reverse order

• Change medium with fresh culture medium containing FBS but no antibiotics (5mL/10cm dish)
• Add DNA plasmids into 500?l jetPRIME Buffer and mix by brief vortexing
• Add 22 ?l jetPRIME to the diluted DNA mixture directly, and briefly spin down
• Incubate at room temperature for 10 min
• Add the 500 ?l of jetPRIME-DNA mixture onto cells drop-wise
• Replace transfection medium with fresh completely culture medium after 4 hours
• Change medium next day (about 18 hours culture)
• Collect the supernatant on 24 hours and on 48 hours after changing medium
  

Plasmids: pCMV-DR8.2 (1?g/?l), pCMV-VSVG (1?g/?l), transfer plasmid (40ng/?l)
Transfection reagent: Lipofectamine 2000

Virus harvesting medium:

• DMEM + 10% FBS + 1g/ml BSA + 1% P/S
• 500 ml Hyclone DMEM high glucose
• 50 ml FBS (Hyclone SH30071.03)
• 64 ml BSA (10g/100ml in DMEM, Hyclone BSA, SH3057402)
• 6ml P/S (antibiotics)

Continually pass 293T cells in 1:2 for three times before seeding the cells into 96-well plate; 

Day 0

• Dilute the cells to a final concentration of 5*105 cells/ml in antibiotics-free culture medium
• Seeding 100?l 293T cells into each well of 96-well plate (i.e., 5*104 cells/well)
• Incubate the cells for overnight (about 16~17 hours) at tissue culture incubator

Day 1

• Dilute the transfer plasmid to a final concentration of 25 ng/?l
• Transfer 4 ?l of each transfer plasmid to the corresponding well in a new 96-well plate
• Prepare packing plasmid DNA (pCMV-DR8.2 + pCMV-VSVG) mixture as shown below; Add 11ug (pCMV-DR8.2), 1.1ug (pCMV-VSVG), 2.31 ml OPTI MEM to a 5ml-tube; mix well; add 21?l to each well of the above new 96-well plate
• Dilute lipofectamine 2000 with OPTI MEM; add 55 ?l lipofectamine 2000 to 2.75 ml OPTI medium; mix well; incubate at room temperature for 5 min
• Add 25 ?l to each well of the new 96-well plate containing plasmids
• Incubate at room temperature for 20 min
• Transfer 50 ?l of DNA-lipofectamine mixture to the cells in each well of 96-well plate
• Centrifuge the cells at 2000 rpm (about 890xg) for 30 min at room temperature
• Incubate the cells at incubator for overnight
  

Day 2

• Carefully remove the culture medium (~120 ?l) from each well after 18 hours culture
• Add 200 ?l BSA-containing DMEM medium to each well
• Incubate the cells at tissue culture incubator for 48 hours

Day 4

• Spin down the cells at 2000rpm for 10 min
• Carefully transfer 190 ?l of culture medium from each well into the corresponding new well of a new 96-well plate
• Pool 5 ?l from each well together for later virus titer
• Seal the plate with sterile sealing film and label the plate
• Store the plate at -80 degrees C before cell infection

4¡ÁLentivirus Concentrator Solution

Dissolve 80g PEG-8000, 14.0g NaCl in 80ml MillQ water and 20ml of 10¡ÁPBS (pH7.4), Mix with gentle stirring, heating gently if necessary, until the solids are dissolved then adjust pH to 7.0~7.2 and the final volume to 200ml. Sterilize by filtering through 0.2?M. The concentrations of PEG-8000 and NaCl in the stock solution are 40% (W/V) and 1.2M, respectively. Store the solution at 4 degrees C.

Virus concentration protocol [See Note 1]

1.  Collect supernatant from 10-cm culture dish into a 15 ml tube; 
2. Spin down at 800¡Ág for 10 min at room temperature or through a sterile 0.45?m syringe filter (e.g. Millipore # SLHV033RS); 
3. Carefully transfer the supernatant into a new 50 ml tube
4. Add 1 volume of concentrator to 3 volumes of supernatant; [See Note 2] 
5. Mix well by shaking for 60sec then incubate with constant rocking at around 60 RPM for at least 4 hours at 4 degrees C; [See Note 3] 
6. Spin down at 1600¡Á g for 60 min at 4 degrees C
7. Carefully remove supernatant without disturbing the pellet; [See Note 4] 
8. Thoroughly resuspend the viral pellet into PBS or desired medium (no serum & antibiotics) with 1/10~1/20 of the original volume by gently pipetting up and down [See Note 5] 
9. Aliquot and store at -80 degrees C until use

Notes

1. The PEG purification step leads to little loss in the infectious virus. However, up to 90% of the input infectious virus can be recovered after PEG precipitation. 
2. The final concentrations for PEG-8000 and NaCl are 10% (w/v) and 0.3M, respectively; Virus is quite stable in PEG solution and can be kept overnight at 4 degrees C without significant loss in titers. Serum in cell culture supernatant may enhance virus precipitation; if you collect the virus in serum-free medium or low-serum medium, or the sample is free of turbidity/small particulates after addition of PEG-8000 concentrator, the addition of sterilized BSA (3% final concentration) may enhance virus precipitation. 
3. Longer incubation e.g. overnight will enhance recovery; PEG will be pelleted with the virus. There may also be other proteins/particulates co-precipitated. It may be necessary to perform a virus purification step depending on downstream application. Residual PEG present in the concentrated virus does not seem to cause any toxicity in culture cells and PEG-purified virus generally is more stable during storage than crude vector preparations
4. The pellet size is not necessarily well correlated with the virus yield. Actually, the pellet you see after centrifugation is not virus only, and most of it is the serum proteins that get spun down from the media. There may also be some genomic DNA from disrupted cells, which does not go into solution, and PEG. To remove the serum protein, thoroughly re-suspend the "pellet" in DMEM without serum or PBS by letting it sit for 10 minutes, then gently pipetting up and down 20-30 times to avoid bubbles, which may inactivate virus. Then transfer this into a microfuge tube and spin in a microfuge full speed for 3 minutes to pellet the protein debris. Aliquot the supernatant and store in a -80 degree C freezer. 
5. If no pellet can be visualized, pipette up and down in the area (and just beyond) where the pellet would form.

References

• Lo HL, Yee JK. Production of Vesicular Stomatitis Virus G Glycoprotein (VSV-G) Pseudotyped Retroviral Vectors. In Current Protocols in Human Genetics, 2007 John Wiley & Sons, Inc. pp 12.7.1-12.7.11
• Lentiviral Vector Preparation Application Note. Beckman Coulter. DS-18017A
• Kutner RH, Zhang XY, Reiser J. Production, concentration and titration of pseudotyped HIV-1-based lentivirus vectors. Nature protocols 2009, 4: 495-905.
• Protocol-Qualitative detection of norovirus and hepatitis A virus in soft fruit.
• Winter VomitingLab: protocols/PEG precipitation
• Sanyal D, Kudesia G, Corbitt G. Comparison of ultracentrifugation and polyethylene glycol precipitation for concentration of hepatitis B virus (HBV) DNA for molecular hybridization tests and the relationship of HBV-DNA to HBe antigen and anti-HBe status. J Med Microbiol 1991, 35: 291-293.
• Colombet J, Sime-Ngando T. Use of PEG, polyethylene glycol, to characterize the diversity of environmental viruses.
• Marino MP, Luce MJ, Reiser J. Small- to large-scale production of lentivirus vectors in lentivirus gene engineering protocols (edited by Federico M) 2003, Humana Press Inc. pp. 43-56

Titering of virus in a 96-well plate format 

1. The day before transduction, seed a 96-well tissue culture plate with HKK293T cells at 2.5~3?104 cells/well in 100ml of growth medium i.e. DMEM with 10%FBS and 1% P/S; [prepare 2.5~3?105 cells/ml to 100ml/well to leave the plate for 1 hr to let the cells attach]
2. 24 hours later, make 5-fold serial dilution of viral stock in a round bottom 96-well plate using serum-free media as shown; [mix the dilution by pipetting contents of well up and down for 10~15 times and discard pipette tips]
3. Gently remove the culture medium from each well, add 30 ml of diluted virus to each well; Spin down at 2000rpm (room temperature) for 2 hrs; incubate the plate at 37 degres C for 4~6 hrs
4. 4. 4~6 hrs later, add 170 ml of growth medium to each well (total 200 ml/well), continue to incubate the cells at 37 degrees C for 3 days
5. 72 hrs later, count the GFP expressing cells or colonies of cells with GFP expression by fluorescence microscopy
6. Calculate the Transducing Units per mL (TU/mL) using the following formula: # of GFP colonies counted ? dilution factor ? 33.3 = # TU/mL (Note: 1000/3=33.3)

Lentiviral Titer by Limiting Dilution-Colony Counting 

Materials

Solutions

1. Puromycin (2mg/ml)
2. 0.1% Crystal Violet Solution (Dissolve 100mg Crystal Violet into 95 ml of MilliQ water plus 5ml of Ethanol);
3. 4% paraformaldehyde

Lentiviral Particles: Packaged lentivirus stored at -80 degrees C

Media and Cells: DMEM; HeLa Cells

Methods

1. Seed 1.5¡Á105 cells to each well of 6-well plate in DMEM medium containing 10% FBS without no antibiotics
2. Incubate the cells overnight at 37 degrees C, 5% CO₂
3. On the next day, thaw lentivirus on ice
4. Remove culture medium from each well and add 975 ?l fresh medium without FBS and antibiotics
5. Perform 5-fold serial dilution of lentivirus
6. Gently add 25 ?l of diluted lentivirus to the cells in each well of 6-well plate
7. Mix by gently turn the plate from one side to another side
8. Optional: Centrifuge at 2000 RPM for 2 hours at room temperature
9. Incubate the cells at 37 degrees C, 5% CO₂ for 6 hours
10. Add 2 ml of DMEM containing 10% FBS and antibiotics
11. Incubate at 37 degrees C, 5% CO₂ for 42 hours
12. Change medium with fresh DMEM containing 2 ug/ml puromycin, 10% FBS and antibiotics;
13. Continue to incubate at 37 degrees C, 5% CO₂ for 4~6 days, and replace culture medium every other day with puromycin-containing medium
14. Observe the cells every day to monitor the death of cells that are sensitive to puromycin
15. Remove the culture medium from each well;
16. Gently add 1 ml of PBS to wash the cells;
17. Fix cells with 1ml of 4% paraformaldehyde for 15 min at room temperature
18. Wash one time with 1 ml of PBS
19. Stain cells with 1 ml of crystal violet solution at room temperature for 20 minutes
20. Remove crystal violet solution
21. Wash cells with PBS for three times (3 ml for each time);
22. Count the blue-stained colonies using a microscope at a magnification of 40¡Á
23. Calculate the lentiviral titer using the formula: Titer = colony number per well¡Ádilution fold ¡Á 40 (TU/ml)