Research and Development on Lentivectors for:
Production of lentivectors can be achieved by transient transfection of the plasmid set into 293T cells by the calcium phosphate method (as shown in the diagram below), or from stable producer cell lines. Some good HIV1-SIN lentivector packaging cell lines exist (see Throm et al., 2009). However, unless very large amounts of a same vector are needed on a regular basis, transient production is still the method of choice for research purposes.
The producer cells
The cells (293T/17 from ATCC Cat# SD-3515) are probably the most critical factor for good titers. They need to be passaged every 2-3 days, otherwise they start to form clumps that cannot be dissociated with one round of trypsin.
Preparation of Solutions
CaCl2, 2H2O (MW 147) SigmaUltra C5080 18.4 g
H2O (milliQ 18.2 Mohm grade) for 250 ml
Hepes 1 M ph 7.3 (Gibco-BRL, Ref 15630-056) 2.5 ml
Filter on 0.22 µm filter. Store at -70°C in 25 ml aliquotes. Once thawed, I store my CaCl2 solution for several weeks (up to 3 months without observing any change in the transfection efficiency).
NaCl (MW 58.44) FLUKA Ultra 71376 8.18 g (0.28 M final)
HEPES (MW 238.3) SigmaUltra H7523 6 g (0.05M final)
Na2HPO4, anhydrous (MW 141) SigmaUltra S7907 0.107 g
H2O (milliQ 18.2 Mohm grade) for 400 ml
Adjust pH to 7.15 with NaOH 10 M solution Sigma BioUltra 72068. Usually, 760 ul are OK. Be careful, pH is VERY important.
Then add H2O to 500ml, and make the final pH adjustment.
Filter on 0.22 µm filter. Store at -70°C in 50 ml aliquotes. Once thawed, I store my HBS solution for several weeks (up to 3 months without observing any change in the transfection efficiency).
H2O (milliQ 18.2 Mohm grade) 25 ml
Hepes 1M ph 7.3 (Gibco-BRL, Ref 15630-056) 250 µl
Store in the fridge. I found that precipitate may not form sometimes. Most times, it is because the pH of the final mix is below 6.9. Check your water and the ventilation of your culture room. To make sure that the solutions can handle "toxic" environments, I use Hepes ph 7.3 in both CaCL2 and water.
Important: Plasmids containing retroviral and lentiviral long terminal repeats (LTRs) are
prone to undergo mutation/deletion in some Escherichia coli strains, such as DH5. After several frustrating experiences using other strains, we now recommend to use STBL3 strain to clone and amplify all lentivector plasmids as well as the psPAX2 packaging plasmid. The same recommendations are also applied by reference plasmid suppliers such as Addgene. We also recommend CcdB Survival 2 T1R strain (Invitrogen) for Gateway® Clonings.
The best DNA is the one you get from JetStar.
In any case, the last step of the DNA prep should be an additional precipitation with EtOH and resuspension in TE 10/1.
For instance, at the end of the Jetstar protocol, after the isopropanol precipitation (11 ml on 15 ml of eluate), redissolve the pellet before it is dry with 500 µl of TE.
Transfer to a clean eppy and quick spin at max speed in a microfuge to pellet the insoluble particles carried over from the column.
Transfer the supernantant into a clean eppy, add NaCl to 0.2 M final and add 500 µl of isopropanol.
Mix gently then transfer the DNA jellyfish to a new eppy containing 1000 µl of EtOH 75%.
Mix gently to wash the DNA and transfer it to a new empty eppy.
Let dry until no liquid is visible, then resuspend overnight with 500 µl of TE.
Minigel and adjust to 1µg/µl.
Do not treat your DNA with phenol or chloroform or whatever.
Also to avoid salt co-precipitation, never precipitate DNA below 20°C.
Transfection of the cells
293T cells are grown in D10 i.e. DMEMhiGlucose (for example Sigma D6429) supplemented with 10% FCS and 1% Peni-Strep-Gluta (Clonagen P11-103)(100x Peni-Strep is 10'000 Units Peni/ml and 10 mg Strepto/ml). The day before in the morning, seed 293T cells at 1.5 to 2.5 millions (cells must be approximately 50 to 70% confluent on the day of transfection) per 10 cm culture dishes with 10 ml of D10.
On day 0, in the evening, make the precipitate according to the following recipe (for one plate of 10 cm)
* psPAX2 encodes for Gag, Pol, tat and Rev and is thus appropriate for the production of second generation vectors (with wild type 5' LTR) as well as third generation vectors (with chimeric 5' LTR such as pCLX and pCWX vectors)
On day 1 in the morning, check your cells. At this point, you should see a very fine and sandy precipitate all over the plate, except on the cells and in their vicinity for they eat CaPO4/DNA precipitate.
On day 2, harvest the sup, and replace with 10-15 ml of medium as the day before. Spin the sup at 2500 RPM for 10 minutes at +4°C, filter through 0.45 µm and store at +4°C.
On day 3, harvest the sup, spin at 2500 RPM for 10 minutes at +4°C, filter through 0.45 µm and pool with the sup of day 2. At this point, you will have a supernatant containing approximately 106 transducing units (TU) per ml. (as titered on HeLa cells).
Concentration of Lentivectors
If you need a more concentrated lentivector suspension, i.e. lots of LVs in small volumes, you will need to concentrate your vector. For concentration, we now use the PEG-it system (System Biosciences, Cat No LV825A-1-SBI).
Phase contrast photograph of 293T cells.
293T/17 cells are seeded the day before transfection at 1 to 3 millions per 10 cm culture dish. At the time of transfection, cells must have the morphology and the density as shown here and be at 50-70% confluence.
|Lentis Titration by FACS|
|Lentis Titration by qPCR|
|Design of efficient miRNAs|
|BSL-2 Emergency Procedures|
|BSL-2 Decontamination Procedures|
|Decontamination of Liquid Nitrogen Tanks|