Lentivirus and Retrovirus 101: basic biology
Shay/Wright Protocol:
Handling Retroviral or Lentiviral Supernatants
1)Before doing TC involving viral supernatants place a small bucket of bleach inside the TC hood. (Spray the bucket down with ethanol and wipe it off prior to placing it in the hood)
2)Every pipette tip that comes in contact with viral supernatant should be placed in the bleach bucket and left there for at least 20 minutes. Aspirate bleach into the tip to at least the level to which you filled the tip with viral supernatant. (If the tip held 10ml of viral supernatant, you should aspirate at least 10ml of bleach before removing the tip from the pipette-aid and letting the tip sit in the bleach bucket.) The same applies for syringes and filters used with viral supernatant.
3)Vials, conical tubes, cell scrapers, or anything else that has touched viral supernatant should be thrown into the bleach bucket and soaked for 20 minutes.
4)If you are disposing of the viral supernatant, simply pipette the media into the bleach bucket, and then bleach your tip as usual.
5)TC dishes that held cells that made or were being infected with virus should be filled with bleach and left in the sink for at least 20 minutes. (If you had 10ml of media in a 10cm dish put at least 10ml of bleach into the dish)
6)After the 20 minute soak you can dump the bleach into the sink andthrow the plastic in the regular biohazard trash for autoclaving.
Second vs Third Generation Lentivirus:
Second generation viruses use transfection of three plasmids into the packaging cells:
Your lentivirus expression vector, a VSVG plasmid such as PMD2G illustrated below, and a gutted lentivirus expressing gag, coding for the virion main structural proteins, pol,responsible for the retrovirus-specific enzymes, and rev, which endcodes a posttranscriptionalregulator necessary for efficient gag and pol expression.In the third generation system, the rev is on a separate plasmid and thus requires 4 different plasmids. The lentiviruses we got from Geron are third generation, almost all the other lenti’s in the lab are second generation.
Third generation offers maximal biosafety but is morecumbersome. The illustration below are vectors made by Trono.
pCMV-dR8.74psPAX2 (“psPax2”) and the envelope plasmid pMD2.G areavailable from Addgene ( psPax2 = plasmid #12260, pMD2.G plasmid #12259.
Lentiviruses and retrovirus are both large and are partially retained in 0.22 um filters, thus use 0.45 um filters to remove any contaminating cells.
According to the TRC consortium,viral harvest growth media containing 10%serum + 1.1g/mL supplemental BSA is equivalent to viral harvest media containing 30% serum – both produce viral stocks with similar high titer (2x higher than in 10% serum). The BSA-supplemented media is more cost effective, easier to mix in standard 500mL media bottles, and may be preferred when transfecting cells that are sensitive to serum.
The half-life at 37C for both lenti- and retroviruses is about 4-6 hours, so one typically collects supernatants twice a day—e.g., first thing in the morning, and last thing at the end of the day.
Matt Porteus showed that including caffeine (to block DNA damage signaling and checkpoint activities) during lentiviralproduction increased titers 2-4-fold. 2,4, or 8 mM caffeine is added the morning after transfection (17 hours) and supernatant collected (in the presence of caffeine each time) at 48 hours and mornings/evenings thereafter. According to JY’s tests, 2mM caffeine was the best compromise between toxicity and increased titer. He made a 40 mM stock (20x) in Medium X, filter sterilized, and added it ~12 hours after transfection. No tests have been done on retroviral titers.
Ecotropic refers to a virus that has the surface env protein that will bind to mouse but not human cells, while amphotropic refers to an envelope protein that will bind to both. An amphotropic packaging cell line expresses lots of the amphotropicenv, and is resistant to infection by a retrovirus expressing the amphotropicenv since all of its receptors are already tied up by the ligand it is already making. Retroviruses sometimes get rearranged following transfection into packaging cells, producing some non-infectious particles. These can be minimized by first transfecting the retrovirus proviral plasmid into an ecotropic packaging line, and then using the supernatant to infect an amphotropic packaging line. Only competent particles can infect the amphotropic line, and this thus eliminates all of the rearrangements so that one ends up with higher titers.
We have both transient and stable systems for the production of retroviral supernatants. In general, a retrovirus that is for a special purpose and isn’t going to be used over and over is most conveniently just packaged in the transient line PhoenixA (A= amphotropic). Routinely used viruses are more conveniently produced in stable packaging lines. For this, first transfect into the transient packaging line PhoenixE (E=ecotropic), and then take the supernatant and use it to infect the stable amphotropic packaging line PA317. After selection using the appropriate drug, the stable line is easily expanded to produce as much supernatant as one wants and can be frozen down and later thawed for additional supernatant production. PE501 is the name of our stable ecotropic retroviral packaging line, but we rarely use it since the transient PhoenixE takes less time.
IMPORTANT: retroviral packaging lines are immortal but they LOOSE expression of their packaging factors over time. We have reselected for retention of the factors (a mess, since diphtheria toxin resistance is one of the selection markers) and designated the reselected cells PD 0. BE SURE to keep track of PDs after thawing the cells and don’t keep them in culture for more than 60-90 PDs. Refreeze low passage cells after thawing them.
The VSVG plasmid expressing the ligand for lentiviral packaging is toxic to cells, so stable packaging lines for lentiviral production aren’t readily available (some attempts to use an inducible VSVG have been done).
Hela cells are somewhat resistant to retroviral infection, perhaps because they are infected with HPV. The env protein from HPV may be binding the receptor and making it unavailable. Several approaches around this include: 1) co-transfecting the proviral DNA into Phoenix A along with a VSVG env plasmid (from the lenti systems). VSVG uses a different cellular receptor, and thus gets in; 2)treating Hela transiently with tunicamycin, to block processing of potential endogenous env proteins. We actually haven’t tried this, and someone should at some point based on J.Virol 1992.66.78-42. They pretreated CHO cells with 0.1-0.3 ug/ml tunicamycin for 19 hours prior to infection with viral supernatants.
Retroviruses only infect dividing cells, and a good retroviral supernatant can infect 10%-50% of the cells. Infecting the cells several times is OK. Lentiviruses can integrate into non-dividing cells, and much higher titers can be produced. The tables below indicate the relationship between the number of cells that are GFP+ after infection with a GFP-lentivirus and the number of integrations per cell. The multiplicity-of-infection (MOI) refers to the number of infectious particles per cell. If one wants only a single integrant per cell, using an MOI such that <30% of the cells turn green is the best approach. For many situations, a high MOI is fine.
Shay/Wright retroviral protocol 2006
Retroviral Protocols
Discard pipets, dishes into 50% bleach
Treat retroviral supernatants with respect but not panic: the half-life of the virus at 37 degrees is about 4 hours.
hTERT is not an oncogene–it does not confer growth advantages by itself.
pBabepuro hTERT-PA317 cells should be resistant to 3 ug/ml puromycin. We do not normally maintain them in puro.
Wear gloves etc.
To harvest supernatants:
Grow packaging cells to near confluence or confluence. Replace medium with “minimal” amount of fresh medium to adequately cover the dish (e.g., 12-15 ml for a 15 cm dish with 150 square cm of surface area).
Harvest medium after 6-8 hours or after overnight, and replace with fresh medium for the next harvest. Harvesting can be repeated twice/day, often for 3-4 days, until the cell layer detaches or the medium gets acidic too quickly. The more confluent the cells, the higher the viral titers will be. Supernatants should be filtered through a 0.45 micron sterile filter, and can be used fresh or frozen and stored for up to about six months at -80 degrees. Freezing causes as loss of about 50% of infectivity, but since the infectivity is usually much more than needed for most tissue culture applications it doesn’t matter.
Convenient method: Harvest medium with a 12 ml syringe (no need to use a needle-just shake off the clinging drop back into the dish) and attach the syringe to a 0.45 micron filter, filter it into a 15 ml tube etc.
To infect cells:
Make viral supernatants 4 micrograms/ml with polybrene (some cells are sensitive and need 2 ug/ml polybrene). If needed, volumes can be increased (if need to infect multiple dishes, or large dishes etc.) by diluting the viral supernatant 1:1 with polybrene-containing fresh medium.
Replace medium of growing cells (retroviruses only infect dividing cells, thus cells can’t be confluent) with enough viral supernatant to cover. Leave on at least four hours: anything more than 8 hours is probably a waste of time. If needed, one can infect first thing in the morning, then again overnight at the end of the day. One infection is usually enough, but can do 2-3 times if a cell type proves hard to infect. After a 36-48 hour expression period, put the cells under selection (puro, neo, etc.).
Sample Retroviral Production Protocol—de Lange lab:
Required Solutions
Polybrene
Stock is 10,000x in medium stored at 4C (40 mg/ml) and user stock is 100x.Final concentration is 4 μg/ml.
Medium for Phoenix cells:
DMEM, 10% FBS, 1% Non-essential amino acids, 1% Pen-Strep, 1% GlutamateStore medium at 4C, warm to 37C before use.
2.5 M CaCl2
Filter sterilize and store aliquots at -20C
2x HBS
50 mM HEPES pH 7.05
10 mMKCl
12 mM Dextrose
280 mMNaCl
1.5 mM Na2HPO4 (FW 141.96)
The final pH of the solution should be 7.05 +/- 0.05. Filter through a 0.2 μm filter, aliquot,and store at -20°C. Try to avoid multiple freeze/thaw cycles. To thaw, warm to roomtemperature and invert or vortex the tube to achieve uniform mixing. Although it isunclear why this occurs, the ability of the 2x HBS solution to produce working CaPO4precipitates deteriorates after 6 months to one year, even when the 2x HBS solution isstored at -20°C.
Bleach
All materials that have been in contact with the retrovirus must be bleached thoroughlybefore discarding in the biohazard waste.
Virus production
Day 0 Plate 2.5 x 106 Phoenix packaging cells in 9 ml medium/10 cm dish in theafternoon. Note: It is very important to have good single cellssuspensions (trypsinize well) and to evenly distribute the cells.
Day 1 Transfect cells with 20 g DNA (±24 hrs after plating), using CaPO4precipitation.Note: At the moment of transfection the cell density shouldbe ± 40-50% such that the cells will be about 90% confluent at Day 3 andcompletely confluent at Day 4.1. In a 2 ml eppendorf tube mix:
50 ul of 2.5 M CaCl2
20 ug DNA (Qiagen prep purified)
MQ up to total of 500 ul
2. While vortexing the tube, slowly add 500 l 2x HBS drop by drop.
Note: if more of the same transfection: mix in 15 ml tube (4 max).
3. Add the 1 ml mix drop by drop to the cells in medium and evenlydistribute by swirling the plate.
4. Place cells back in incubator.
Day 2 Change medium 5-20 hours after transfection (precipitate is very fine).Late in the afternoon or in evening replace medium with 9 ml of freshmedium. Note: Phoenix cells detach easily, be careful with all mediumchanges.
Day 3 Collect first supernatant (T1) late in the afternoon (this is 48 hrs aftertransfection and not later than 24 hrs after changing medium):
1. Remove virus-containing medium and set aside for a moment tosupply the packaging cells with 9 mls of fresh medium (1-2 platesat a time).
2. Filter the virus-containing medium through a 0.45 um filter andimmediately use for infection.
Day 4 Collect second supernatant (T2) in the morning.
Collect third supernatant (T3) in the evening (8 hrs after T2).
Lentiviral Packaging Protocols
Below are a whole series of different protocols. Things to consider in using/designing your own protocol: including caffeine can increase titers 2-4x; some protocols claim higher titers in the absence of antibiotics; some protocols claim higher titers using 30% serum or 10% serum + 1000 mg/ml BSA (Note: JY claimed higher titers with lowering serum to 5%); for large scale production (e.g., many 15 cm plates for libraries) consider using CaPO4ppt transfection protocols. I don’t know if anyone has tested combining caffeine, antibiotic free, and high protein.
Polyjet protocol from Guido Stadler 2011
Transfection of 293 cells with PolyJet (Signagen# SL10068B)
seed 800k (6 well) / 5M (10cm) 24 h before transfection in gelatin-coated dishes (gelatin prevents 293 cells from detaching when they become over-confluent during virus-harvest; gelatin is not needed for routine culture of 293 cells. Myoblast group routinely coats plates with gelatin, it’s easy)
change medium to 1/5 ml fresh growth medium 30-60 min before transfection
dilute 1/5 ug DNA into 50/250 ulmedium X, vortex gently and spin down
Lentivirus production: in 6 well dishes: 0.75 ug target plasmid, 0.75 ug helper plasmids (equimolar ratio of helper plasmids is recommended: ~0.49 ug psPAX2, ~0.26 ug pMD2G)
dilute 3/15 ulpoly jet into 50/250 ulmedium X, vortex gently and spin down
add diluted poly jet to the diluted DNA (not the other way round!), vortex and spin down, incubate 15 min (not longer than 20) at RT
add the mixture drop-wise onto the medium in the dish, mix by swirling the plate
changemedium 12-18 h after transfection (5 h for sensitive cells)
use 2mM caffeine in medium for virus production
caffeine stock solution: dissolve 40mM in medium X, filter (MW=194.19 g/mol; 7.77 g/l (388.4 mg/50ml) = 40mM)
Effectene protocol from Ugur Eskiocak~2010
Transient Transfection of 293FT cells using Effectenefor virus production (6 wellformat)
Protocol by UgurEskiocak
1.The day oftransfection, dilute 1 ug DNA (0.5 ug vector + 0.2 ug PMD2G + 0.3 ugPsPAX2) in 100 ul(total volume) Buffer EC.
2. Add 8 ul Enhancer and mix by pipetting up and down. Incubate at room temperaturefor 2-5 min.
3. Add 8 ulEffectene to the DNA-Enhancer mixture. Mix by pipetting up and down.
Incubate the samples for 5-10 min at room temperature to allow transfection-complexformation.
4. While complex formation takes place, wash, trypsinize and collect cells in a 15mlcentrifuge tube. Count the cell numbers.
5. Add 600 ul growth medium (can contain serum and antibiotics) to the tube containing the transfection complexes. Mix by pipetting up and down, and immediately add thetransfection complexes drop-wise onto well.
6. Plate 2M cells/well on the top of transfection mix in the 6 well.
7. Incubate the cells with the transfection complexes under their normal growthconditions and collect viral soups 24, 48, and 72 hours after transfection.
Effectene protocol from Ryan Laranger for Myoblasts ~2010
MAKING "hCPK4 VIRUS" FOR TRANSDUCTION (in 293FT in MediaX 10%S)
PMD26 = 285ng/ul PSPAX2 = 345ng/ul pUE3-lenti-hCDK4 = 250ng/ul
l.MIXIN A TOTAL Of lug of DNA contained in 100ul of Buffer EC:
0.5 ugof~hCDK4 vector (2 ul)
0.2 ug of PMD2G (1 ul)
0.3 ug of PsPAX2 (1 ul)
2. • Add 8 ul of Enhancer and mix by pipetting upand down.
• Incubate at room temp for 2-5 minutes
3.•Add 15 ul of Effectene to the DNA-Enhancer mixture.
• Mix by pipetting up and down.
• Incubate for5-10 f inutes at RT
4.Wash, trypsinize and collect cells in a 15ml falcon tube. Count the cell and dilute them to lmillion/ml
5. Add 600 ulof growth mediumto the tube containing the transfectioncomplexes.
Pipette up and down and immediately add the transfection complexes drop-wise onto wells
6.•Plate2 million cells per well on top of the transfection mix (6 well tray)
7.•Incubate cells with the transfection complexes and collect viral sups
(replacel.5ml of media; freeze down 750ul and use 750ul to transduce).
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Lentiviral Production Protocol adapted from Marculab, obtained from JY Kim~2009
-The day before transfection:
I. Plate out __ 293FT cells per 10cm plate or __ (up to 6M) cells per I5cm plate
ineither 10 or 25ml X media, respectively.
-The day of transfection:
1. Check to make sure cells are at least 25% confluent before continuing.
2. Combine the appropriate amount of plasmid DNAs in a 15ml conical tube:
For 10cm plates:
5ug DNA
3-4ug psPAX2. (structuralvector)
1-2ug pMDG2 (envelope vector)
10ugTOTAL
For I5cm plates:
I5ug DNA
7.5ug psPAX2 (structural vector)
7.5ug pMD2.G (envelope vector)
30ugTOTAL
(rest of the protocol is for 15cm plates)
3. Add filtered water to a final volume of 878ul. )
4.Add 122u12M CaCl2, (stock in -20C walk-in freezer) and mix.
5. Add 1000ul of2xHBS (stock in -20C walk-in freezer).
6. Use a 1 ml pipette tip and an automated pipettorto generate bubbles for l0s at thebottom of the tube and l0s going up and down the mixed solution.
7. Let mixture sit for 15m.
8. Remove appropriate amount of media from 293FT cells so that only 18ml is left.
9. Add all of mixture to the cells gently and evenly. Swirl to distribute evenly and putback in incubator OIN.
-The day after transfection:
1. Check transfection efficiency for fluorescent marker if possible.
2. Change to the media for the cell type for which the virus is being produced.
-The next day:
1. Harvest viral supematant and filter through a 0.45Um filter.
(can collect viral supernatant up to 3 days after transfection)
2xHBS:50mM HEPES
10mM KCl
12mM Dextrose
280mM NaCl
1.5mM Na2HPO4
Resuspend in 900 ml H2O, adjust pH to 7.04 (with NaOH if using HEPES free acid, with HCl if using HEPES sodium salt). Raise volume to 1000ml and carefully re-adjust final pH to 7.05 exactly (IMPORTANT: pH needs to be very precise at pH 7.04 – 7.05). Filter through 0.22 uM and store in aliquots at -20°C (should be good for up to 6 months).
The RNAi Consortium Protocol
1/18/07
Section II: Lentiviral Production
Introduction:
This section contains protocols for the production of lentivirus stocks from hairpin-pLKO.1 plasmids in 6 cm plates. Lentiviral production consists of the following steps:
Day 0 Seed 293T packaging cells