Stable cell line generation via retrovirus

Day 0: Thaw fresh cryovial of cells to be transduced d

Thaw ~ 1 week prior to planned transduction day, use

low passage number if possible. Aim for cells to be 80% confluent on day of transduction.

Recommend seeding four wells of a 12-well plate (100,000 cells/well) the day

before transduction (after collecting virus).

Day 1: HEK293T Transfection

1. Prepare 2 80% confluent 10 cm plates of HEK293T for transfection

2. Prepare 3 mL of warm Opti-mem solution. Add 10 µg of retro/lentiviral transfection plasmid, 10 µg VSV-G plasmid, and 10 µg pCMV-MLV (if retroviral)[DT1] [DT2] or 10 µg pCMV R8.74 (if lentiviral)

3. Add 90 µL of LT-1 reagent and swirl.

4. Incubate at room temperature for 15 minutes.

5. Add 1.5 mL dropwise to each 10 cm HEK293T plate.

6. Incubate for 3 days in incubator before harvesting viral particles. If a fluorescent marker was included, it should begin to be visible on days 1,2 post transfection.

SAFETY: After

this point all media and plasticware is to be treated as potentially containing

viral particles. Decontaminate using 10% bleach for 10 minutes before

discarding as red bag waste. Rinse pipettes with 10% bleach before discarding

as biohazard waste.

Day 4: Harvest viral particles

1. Collect media from both HEK293T plates, combine into one 20 mL portion in a 50 mL falcon tube. Bleach and discard used HEK293T plate.

2. Spin 50 mL tube containing harvested viral particles in Sorvall for 2 min at 2,000 rpm[DT3] [DT4] to clarify media and pellet stray cells

3. Aspirate off media without disturbing pellet.

4. Add 6 mL Lenti-X concentrator solution to 18 mL of recovered media (concentrator is 4x). Invert to mix.

5. Incubate in refrigerator 1 H – O/N[DT5]

6. Spin 50 mL conical containing viral particles in centrifuge at 1,500 rcf for 45 min

7. Remove 2 mL of supernatant from top of pelleted solution and save.

8. Aspirate off remaining media to recover viral pellet. Bleach excess media.

9. Resuspend retro/lenti pellet in 2 mL of saved medium.

10. Titrate concentrated retro/lenti solution into prepared 12-well containing target cells. Recommend 100-200-400-800 µL titration for 4 total attempts at transfection. Typically 400 or 800 uL will yield ~100% transfection efficiency

11. Return 12-well plate containing transduced cells to incubator.

Day 5: Check on transduction n

1. May be able to see fluorescence today.

2. Swap media to fresh.

Day 6-7: Check on transduction n

Day 8-:

1. Allow cells to grow until almost confluent on 12-well plate. Trypsinize and replate in a 6-well, allow to grow until confluent, then replate in a 10 cm plate.

2. Split into two 10 cm plates, Freeze one plate down into 4 cryovials the next day to save progress. Label with estimated transduction efficiency if able to measure.

3. Take other plate and select with antibiotics if able to/necessary, or clonally isolate populations if able to/necessary.