Lentivirus production and transduction

Transfection

1.One day before lentivirus production, plate HEK293 cells on 6 cm dish at seeding density 3x10⁶ cells in growth medium (DMEM+10% FBS) without antibiotic. Cells should reach 90% confluency on the next day.

2. Prepare the plasmids mixture and Lipofectamine 2000 mixture as follow:
    • Vector mixture:

    • Lipofectamine mixture:

     Incubate vector and lipofectamine mixture at RT for 5 minutes.

3. Add lipofectamine mixture to the vector mixture drop by drop. Mix gently and incubate for 15 minutes at RT.

4. Wash HEK293 cells with Opti-MEM.

5. Add 1.5 mL Opti-MEM to the cells.

6. Add the vector and lipofectamine mixture to the cells drop by drop. Incubate the cells at 37°C for 6 hours.

7. Add 2 mL growth medium without antibiotics to the cells. Incubate cells at 37°C overnight.

8. The next day, change the medium to a fresh growth medium without antibiotics.

Harvesting viral supernatant at 48-96 hours post transfection

1. Viral supernatant was harvested at 48,72,and 96 hours post transfection.
2. Supernatant were collected and centrifuge at 500xg for minutes followed by filtering through 0.45 µm filter. The collected supernatant was pooled together from 48- 96 hours post transfection and stored at 4°C for later use.
    

Concentrate viral supernatant

1. Prepare 4x PEG8000/NaCl solution as follows:
   Dissolve 80gram PEG800, 14gram NaCl in 80 mL in 80 ml water and add 20 mL of 10x PBS pH 7.4. Mix using hotplate stirrer until the solids are completely dissolved and then adjust the volume to 200 mL and adjust the pH to 7.0 – 7.2. The concentration of PEG800 and NaCl in this stock solution are 40% (w/v) PEG8000 and 1.2 M NaCl, respectively. Sterilize by filtering through 0.2 µm and store at 4°C.
2. Add 1 volume of PEG8000/NaCl solution to 3 volumes of viral supernatant.
3. Incubate the mixtures for 16-20 hours on a shaker at 4°C.
4. Centrifuge at 10.0000x g for 30 minutes at 4°C.
5. Remove the supernatant and re-suspend the viral pellet in sterile PBS or DMEM with 1/10 or  1/20 of the original volume by pipetting up and down.
6. Measure virus titer using the QuickTiter™ HIV Lentivirus Quantitation Kit (HIV p24 ELISA) according to the manufacturer instructions.
7. Aliquot concentrated lentivirus and stored at -80°C until use.

Lentivirus transduction protocol:

1. Add concentrated lentivirus to reach final concentration 10⁶ TU (Transducing Unit)/mL in the appropriate cell target medium containing 6 µg/mL polybrene.
2. Incubate cells at 37°C overnight.
3. The next day, change to fresh media.