Source: M. Scott (9/93)
Reference: Pear, W. S., Nolan, G. P., Scott, M. L., and Baltimore, D.
"Production of high-titer helper-free retroviruses by transient transfection."
Proceedings of the National Academy of Sciences 90: 8392-8396 (1993)
Reagents*
1) 2x HBS
Reagent Final [] grams dry
HEPES, pH 7.05 50 mM 5.0
KCl 10 mM 0.37
dextrose 12 mM 1.0
NaCl 280 mM 8.0
Na2HPO4 (FW 141.96) 1.5 mM 0.1065 g
500 ml total, pH to 7.05
2) CaCl2
CaCl2 2 M 29.4 g/ 100 ml
*stock solutions may be prepared and frozen at -20 C
Vtotal (ml) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
2xHBS (ml) 0.25 0.5 0.75 1.0 1.25 1.5 1.75 2.0 2.25
CaCl2 (µl) 31 62 93 124 155 186 217 248 279
H2O (µl) 219 438 657 876 1095 1314 1533 1752 1971
Procedure
1) 293 cells are grown in Dulbecco's minimal essential medium supplemented
with 10% IFS and penicillin/streptomycin. Be sure to use DME and not other media--with
incorrect bicarbonate concentrations, an orange juice-like precipitate will
form in 1-3 hours after addition of DNA. Cells are seeded at 2 x 106 cells/60
mm plate in a volume of 3 ml of medium the night before transfection.
.
2) For a 60 mm plate, 10-25 µg replication-defective virus DNA
is used in a Vtotal of 0.5 ml. Increasing amounts of DNA tend to yield higher
titers up to about 20-30 µg per plate.
a) DNA's should be clean (for plasmids, twice banded in CsCl is what
I prefer; others have found preparations from Quiagen columns satisfactory,
too).
b) the amount of DNA the cells will tolerate depends upon the construct;
toxic genes such as bcr-abl may require smaller amounts of DNA.
3) Mix H20/DNA and CaCl2; add to this the 2x HEPES with bubbling using automatic pipettor for 15 seconds.
4) Add the ppt thus formed (should be slightly cloudy) immediately to tissue culture cells; I usually use a plastic pipette and add the precipitate dropwise. A mild-moderate drop in pH of the medium is normal. Place cells immediately into incubator and incubate overnight.
5) Change the medium the day following the transfection; 24 hours after the medium change (about 48 hours after addition of DNA), harvest supernatants. If the cells were transfected with a replication competent retroviral construct, filter as usual and treat as a usual retroviral stock. Titer tends to drop after about 60-72 hours after addition of DNA. The cells are very confluent at virus harvest, but should remain attached; the surface of the medium may appear slightly oily at this time.
All conditions are for 60 mm plates; scale-up to 100 mm plates may or may not
be equally efficient, but works well enough for many purposes
1. The cells are carried in gpt selective media prior to freezing. To maintain retrovirus production, one can grow in selection medium, freeze, then thaw cells monthly to be grown in DME/10% FCS/PS
Selection medium:
415 mls DME
50 mls dialyzed FBS
5 mls 100x PCN/STREP (Gibco)
10 mls 50x HAT media (Sigma)
20 mls 25x xanthine (5 mg/ml stock)
5 mls L-glutamine
5 mls 100x mycophenolic acid (2.5 mg/ml stock)
2. Plate 2 x 106 cells/plate approx 18-24 hrs prior to transfection in 4 mls of DME/10% FCS/PCN/STREP without selection.
3. Just prior to transfection, change media to 4 mls of DME/10% FCS/PCN/STREP containing 25 µg/ml chloroquine. WIthin 5-10 minutes, add DNA as below.
4. Transfect using 6-10 µg DNA in CaCl2/H2O in a volume of 500 ml. Add 500 ml 2 X HBS by bubbling. Immediately (within 1-2 minutes) add this solution to the cells.
5. At 10 hours, suck off the media and replace with 4 mls 10% FCS without selection. 24 hours prior to harvest, you may want to change volume of media to 2.5-3 mls in order to increase titer.
6. The cells should be nearly confluent by 24 hrs after transfection. I harvest the cells at 48 hours post-transfection. If the cells are not confluent at this point, you may want to wait until 72 hrs. If the cells are not confluent at this time, you should play with the conditions so that they are confluent by 48 hours.
To infect:
8. Plate 5 x 105 3T3s the night prior to infection on a 100 mm plate.
9. Suck off supe from TRANSFECTED PLATES and spin 5 min at 1500 x g to remove cells (temp is unimportant).
10. Add virus containing 4 mg/ml Polybrene such that the final volume is 3 mls. Freeze the rest of the viral supe at -80 C.
11. Suck off media from 3T3s and pour on the 3 mls containing virus and Polybrene. Leave on cells from 3-5 hours (although the cells can survive even longer without loss of titer). After this time, suck off supe and replace with 10 mls DME/10% CS/PCN/STREP.
12. Harvest (neo select, etc) these cells at 48 hours.