Lentivirus generation and MCF10A transduction

Lentivirus generation and MCF10A transduction

Joanna Y. Lee | Chaudhuri Lab

Dec. 2018

Materials:

HEK 293T cells

293T growth medium

500 ml high glucose DMEM (cat. #11995065; Gibco)

10% FBS (cat. #SH30071.3; Hyclone)

5 ml 100x GlutaMAX (cat. #35050061; Gibco)

5 ml 100x penicillin/streptomycin (cat. #15140; Invitrogen)

Opti-MEM (cat. #31985062; Gibco)

Lipofectamine 3000 (cat. #L3000015; Invitrogen)

Lentiviral packaging plasmid (pCMV-delta-R8.91)

Lentiviral envelope plasmid (pMD2.G-envelope)

Lentiviral transfer plasmid (e.g. GFP, sgRNA)

0.22 µm Steriflip (cat. #SCGP00525; Millipore)

Lentivirus Precipitation Solution (cat. #VC100; Alstem)

Low passage MCF10A cells (cat. #CRL-10317; ATCC)

MCF10A growth medium

Polybrene (cat. #SC134220; Santa Cruz Biotechnology)

Note: all lentiviral waste must be bleached. Bleach solid waste (e.g. pipette tips, tubes) with 10% bleach, add bleach to liquid waste to make 10% final concentration.

Seed cells for transfection:

1. Seed 293T’s to be 70-90% confluent next day: 1 x 107 cells in 10 cm plate (depends on growth rate of cells).

Transfection:

2. (Next day) Only transfect if cells are 70-90% confluent.

3. In TC hood, prepare Lipofectamine and DNA mixes:

1. Tube A:

1. 1.5 mL Opti-MEM

2. 41 µl Lipofectamine 3000

2. Tube B:

1. 1.5 mL Opti-MEM

2. 11 µg packaging plasmid

3. 1.3 µg envelope plasmid

4. 5 µg transfer plasmid

4. Add solution from Tube A into Tube B and mix by pipetting or gentle vortexing. Note: vigorous vortexing will shear plasmid DNA.

5. Incubate 10-20 min at RT to allow lipid-DNA complexes to form.

6. Remove 5 ml media from 10 cm dish of 293T cells.

7. Gently add lipid-DNA complex mix against wall of each plate to avoid dislodging cells. Gently shake plate.

8. Incubate cells for 4-6 h at 37ºC with CO2.

9. Change transfection medium, pipette 10 ml warm media against wall of plate to avoid dislodging cells. Bleach media waste.

10. Incubate at 37ºC with CO2.

11. Harvest 48 h post transfection.

12. (Next day) Check cells for transfection by IF, if transfer vector contains fluorescent marker.

Harvest and concentrate virus:

13. Prewet 0.22 µm Steriflip with 500 µl PBS. Use vacuum trap to filter through.

14. Use 10 ml pipet to add ~8 ml virus containing media from 10 cm dish directly onto Steriflip filter. Filter through.

15. Rinse plate with 1 ml PBS and pipet onto Steriflilp filter. Filter through.

16. Add 2.5 ml cold Lentivirus Precipitation Solution to ~10 ml filtered viral supernatant (1:4).

17. Mix and refrigerate 6 h up to 3 days.

18. Centrifuge mixture at 1500 x g for 30 min at 4ºC. After centrifugation lentivirus may appear as beige pellet at bottom of tube.

19. Remove supernatant by pipetting, leaving ~100 µl sup. Bleach waste.

20. Resuspend pellet in remaining ~100 µl sup and transfer to 1.5 ml tube.

21. Remove all traces of fluid by centrifuging for additional 1500 x g for 5 min and removing supernatant by pipetting, making sure not to disturb viral pellet.

22. Resuspend lentiviral pellets in 100 µl serum-free DMEM/F12 (1/100 of original volume).

23. Store at 4ºC for short term or aliquot and store at -80ºC for long term (freeze/thaws will decrease viral infection efficiency).

Seed cells for infection:

24. Seed MCF10A cells to be 40% confluent next day: 2 plates 3 x 106 cells in 60 mm dishes (1 plate for antibiotic kill control)

Infect cells with lentivirus:

25. (Next day) Prepare MCF10A transduction medium:

1. 15 ml complete MCF10A growth media

2. 12 µl 10 mg/ml polybrene

26. Replace medium in each 60 mm dish with 2 ml transduction medium.

27. Add 20 µl concentrated virus (or nothing to kill control plate).

28. Incubate overnight at 37ºC with CO2.

29. (Next day) Change to 5 ml fresh growth medium containing antibiotics (e.g. puromycin), if lentiviral transfer vector contains antibiotic selection marker. Note: polybrene can be toxic to cells with long exposures, >12 h.

30. Check cells over next few days for transduction – either by survival of antibiotic selection (using kill control plate as reference), fluorescence (if lentiviral transfer vector contains fluorescence marker), or both.

1. Transduced MCF10A cells should be visible by fluorescent marker 24 h post infection.

2. Puromycin should kill uninfected cells within 72 h post infection (use kill control as reference).

31. Split cells as necessary to get 2 confluent 15 cm dishes.

32. Freeze down at least 1-15 cm dish of cells into 5 cell stocks; use other plate for Western blot or to begin assaying for phenotype.