Transfection Protocol for Vector
Production in 293T cells
Preparation
of Solutions
CaCl2 0.5 M
CaCl2, 2H2O (MW 147) SigmaUltra C5080 36.75 g
H2O distilled (preferably not MilliQ but you can test) or double-distilled for 500 ml
Store at -70°C in 50 ml aliquotes. Once thawed, I store my CaCl2 solution for several weeks (up to 3 months without observing any change in the transfection efficiency).
HeBS 2x
NaCl (MW 58.44) SigmaUltra S7653 16.36 g (0.28 M final)
HEPES (MW 238.3) SigmaUltra H7523 11.9 g (0.05M final)
Na2HPO4, anhydrous (MW 142) SigmaUltra S7907 0.213 g (1.5 mM final)
H2O distilled (preferably not MilliQ but you can test) or double-distilled for 800 ml
Adjust pH to 7.00 with NaOH solution. Be careful, pH is VERY important. Below 6.95, precipitates won't form, above 7.05, precipitates will be coarse and transfection efficiency pathetic. So double check the calibration of your pHmeter or go hiking instead, at least you will spare the chemicals.
Then add H2O to 1000ml, and make the final pH adjustment.
Store at -70°C in 50 ml aliquotes. Once thawed, I store my HeBS solution for several weeks (up to 3 months without observing any change in the transfection efficiency).
H2O for mixing with the plasmids
H2O distilled (preferably not MilliQ but you can test) or double-distilled 50 ml
Hepes 1M ph 7.3 (Gibco-BRL, Ref 15630-056) 125 µl (2.5 mM final)
I found that the aspect and quality of the precipitates can vary among batches of distilled water. To get around that, I am now buffering my water. With 2.5 mM final of Hepes in the water, it helps maintaining a proper pH and does not compete for the final pH with the HeBS ph 7.00 which is 25 mM final is the mix (Hepes pH 7.3 from the water is 0.625 M final). Store at +4°C.
Tips on DNA quality: The best DNA is the one you get from Qiagen Kits of JetStar (although it varies from batch to batch). Second, close, is from Wizard Promega (or Merlin). 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 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, do not precipitate DNA below
20°C.
Transfection
of the cells
The day before, split the cells at 1 to 3 millions (cells must be approximately 1/4 to 1/3 confluent on the day of transfection) per 10 cm culture dishes with 10 ml of DMEM 10% FCS.
On day 0, in the evening, make the precipitate according to the following recipes (*) (for one plate of 10 cm).
Recipe 1
Mix:
|
|
2nd generation |
3rd generation |
||
|
Envelope plasmid |
pMD2G |
5 µg |
pMD2G |
5 µg |
|
Packaging plasmid |
pCMVdeltaR8.74 |
15 µg |
pMDLg/pRRE |
10 µg |
|
Rev-expression plasmid |
|
|
pRSV-Rev |
5 µg |
|
Transfer vector |
pHR (**) |
20 µg |
pRRL (**) |
20 µg |
Recipe 2
Mix:
|
|
2nd generation |
3rd generation |
||
|
Envelope plasmid |
pMD2G |
3 µg |
pMD2G |
3 µg |
|
Packaging plasmid |
pCMVdeltaR8.74 |
6.5 µg |
pMDLg/pRRE |
5 µg |
|
Rev-expression plasmid |
pRSV-Rev |
2 µg |
pRSV-Rev |
2.5 µg |
|
Transfer vector |
pHR (**) |
10 µg |
pRRL (**) |
10 µg |
* these recipes are the result of long optimizations in our laboratory and
in the laboratory of Luigi Naldini, and discussions with Antonia Follenzi.
** pHR refers to second generation vectors with wild type 5' LTR and pRRL
refers to third generation vectors with chimeric 5' LTR.
Adjust to 250 µl with the special water. Add 250 µl of CaCl2 0.5 M. Mix well and add this mix, DROPWISE, SLOWLY (one drop every other second), on 500 µl of HeBS2x, while vortexing topspeed. Let stand still for 30 minutes minimum (40 minutes max) on the bench. This time is critical for optimal formation of the precipitate. If you wait too long you may have a coarse precipitate, too short, you will form very few precipitate, and once diluted in 10 ml of medium there is no chance to precipitate. Also you may want to incubate your solutions at RT or 37°C before mixing to standardize the precipitate characteristics.
Then add the precipitate slowly, dropwise on the cell monolayer. The plate should then be gently SHAKEN NOT STIRRED, or all the precipitate will be in the center. At this point, you should not see the precipitate, for it is too fine and will take hours to sediment on the cells. If you see it, then it is too coarse and your transfection will most likely be less efficient.
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. Discard the medium after gentle, but firm, stirring (to eliminate the maximum of precipitate), and replace with 10-15 ml of new medium.
Important notes: The medium must be prewarmed at 37°C or else the 293T will have a thermal shock, shrink and detach. You must add the medium very gently to the cells. Even so, you will make a hole in the monolayer.
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 use Beckman Konical tubes (ref 358126). Put 4 ml of sucrose 20% in water on the bottom of the tube and fill up to the top with filtered sup, spin at 26 kRPM for 90 min and discard gently the supernatant by inversion. Let the tube dry inverted and resuspend the pellet (not always visible) with complete medium or serum-free medium (such as CellGro® Stem Cell Growth Medium (SCGM)) if your next experiment requires the absence of serum.
The use of protein-containing medium is preferable over PBS to resuspend the viral pellet for 2 reasons. First, the presence of proteins will protect the viral particles and second, the surfactant effect of proteins will help redissolve the pellet.
Production
of Lentivectors (pdf)