Stock solutions: 1. 2.5 M CaCl2 stored at -20C (10X)

PROTOCOL FOR TRANSFECTION RETRO/LENTIVIRAL PRODUCTION ( VERMA LAB )

Stock solutions: 1. 2.5 M CaCl2 stored at -20C (10X)

2. 0.5 M BES (10X)

3. 150mM Na2HPO4 (100X)

4. 2.8 M NaCl (10X)

5. Sterile filtered ddH20

6. sterile 1N NaOH

For best results make up all solutions the day of transfection. The mixed solution can be stored at 4C for up to a week. Some people store the ready 2XBBS at -20C indefinitely however in these conditions transfection efficiencies may vary.

Mix for 20 ml or 40ml 2X BBS:

14.6 ml ddH20 29.2 ml ddH2O

2.0 ml BES 10X 4.0 ml BES 10X

2.0 ml NaCl 10X 4.0 ml NaCl 10X

0.2 ml Na2HPO4 100X 0.4 ml NaHPO4 100X

0.4 ml 1N NaOH 0.8 ml NaOH (1N)

add 1N NaOH in increments of 10-20 ml until pH is exactly 6.95 and sterile filter with a 0.22m syringe filter and store at 4⁰C for up to a week to 10 days.

Transfection protocol for retroviral/lentiviral production.

1.use 20-30 mg DNA per transfection of a 10 cm dish. (For retroviral production use 5-10 mg VSVG and 10-20 mg of vector in 293 gp cells)(Lentiviral production: 293T cells split 1:5 the night before, use 10 mg of the vector, 10 mg PMDL, 6 mg pMDG(VSV-G), 4 mg RSV-REV)(0.5-1 mg of pEGFP can be added as a tracer for transfection efficiency). Growth medium for the cells can be DMEM of IMDM ( recommended especially for higher cell densities). A typical large prep for in vivo use is approximately 25-100 X 15 cm dishes.

2. add H20 to 400 ml.

3. slowly add100 ml of 2.5M CaCl2 dropwise to DNA solution while vortexing .

4. Add dropwise 0.5 ml of 2X BBS to CaCl2/DNA solution while vortexing as in step 3.

5. Allow precipitate formation for about 5 min, (can be less but not longer).

6. Pipette solution up and down a few times to homogenize precipitates before adding to 10 cm dish.

7. leave cells O/N in 3% CO₂ and change to fresh medium the next day.

At this time successful transfection will lead to most cells on the dish to form syncytia. Move the cells back to a 10% CO₂ incubator.

8. For retroviral production 1^st harvest is 48 hours after change of media and 2nd harvest is 24 hours after that. For Lentiviral production harvest every 24 hrs after change of medium for as long as your vector produces significant amounts of virus (to be determined empirically by p24 ELISA or biological titering). (Store at 4C for up to 72hrs until spin).

9. Harvest by collecting media and filtering it through a 0.45 m filter and freeze at -70C

10. (Optional for in vitro studies) If concentration is desired or required spin down collected media with a sucrose cushion(20%) ~0.5 ml (necessary for in vivo studies only) at 50,000 X g (SW-28 ultracentrifuge rotor or 25.50 superspeed rotor). with 2 spins 40 X 10 cm dishes can be concentrated to about 100 ml. Each spin decreases titer about 50%.

11. Resuspend pellets in PBS or HBSS by pipetting (may take several minutes depending on the virus) and vortexing for at least 30-60 minutes.. The virus prep appears as a milky suspension. Typical titers of viruses prepared in this manner is about 10⁸ to 10¹⁰ transducing units/ml. For in vivo studies to reduce toxicity apply a quick spin and discard the pellet. The virus is then frozen at —70C for long term storage.

Note: cells should be seeded the night before to avoid acidification of the medium. alternatively medium can be changed prior to transfection if feasible. To increase titers and ease of manipulation especially of 293 cells coat the dishes with poly-L lysine 0.01% for at least 1hr. You can also add Na-Butyrate up to 5mM final after changing the medium. Good transfection efficiencies of 293 cells with this method are 80-100% and 20-30% with HeLa.

Rescue of Failed Transfections:

If a transfection does not yield much precipitate it is most likely due to an incorrect pH of the 2XBBS transfection buffer. A failed transfection (one which does not yield much precipitate) can be rescued under certain circumstances i.e. cells not too confluent and failure is due to incorrect pH. To do the following work quickly: take the medium (about 10 ml/10cm dish) in which the DNA and the Calcium are dissolved and pipette into a 50 ml conical tube. While vortexing add 1 volume of RPMI medium. Add the mixed medium back to the dish immediately and place dish with the DMEM/RPMI mix back into the 3% CO₂ incubator immediately. From now on handle the transfections as usual but everything will be delayed a few hours.

GP/ LOG-V 97

updated 3/2000