Retro/Lentivirus production

Producing Retroviral transduced cell lines: - 06/12/13

Day 1:

Add 5 x 10⁵U2OS cells in a 60 mm dish in a volume of about 0.5-1mL. Add 2mL of retrovirus. +Add 4ug/ml final concentration of polybrene. Aim for a total volume of 2.5-3mL. Be sure and include an extra dish of cells with no virus to serve as a selection control.

Day 3:

Split the cells and move all of them to a 100mm dish and add selection media (Puromycin 1-2ug/mL, G418 1ug/mL, etc).

Day 5:

All the cells in the control dish should be dead. Split the antibiotic resistant cells as appropriate. I usually maintain them in selection levels of antibiotic for 2 passages and then bring them down to a maintenance level of half of the selection concentration.

Antibiotic Concentrations for selection:

Hela, U2OS, hTERT-RPE, and HCT-116

1-2ug/mL Puromycin (1-2 days)

1ug/mL G418 (2-4 days)

Kareem Mohni - David Cortez Laboratory

Package and Titer Lentiviruses - Updated   08/07/13

Reagents:

293T cells – for packaging

Hela/U2OS cells – for titration

pLKO.1 (shRNA expressing lentiviral plasmid) or pLPG (protein expression with weak promoter)

psPAX2 – gag/pol expressing plasmid

pMD2.g – VSV-G expressing plasmid

Packaging Lentiviral Particles

Day 1:

Plate 4-5 x 10⁶293T cells in a 100mm dish. Cells should be about 80% confluent on the day of transfection (Day 2).

Day 2:

Transfect cells with 4ug pLKO.1, pLPG, or appropriate lentiviral vector, 3ug psPAX2, and 1ug pMD2.G in 100uL DMEM and add 24uL of 1mg/mL PEI. Incubate for 10-15 minutes at room temperature and add to 10mL of complete media on cells.

Day 3:

Aspirate media, wash cells once in PBS, and add 7mL DMEM + 10% FBS + Pen/Strep.

Day 4:

Collect media in a 15mL tube and place at 4°C. Add another 7mL media to the transfected cells.

Day 5:

Collect media and pool with the media from the day before in the 15mL tube. Spin the collected media at low speed to pellet any cellular debris. Move supernatant to a new 15mL tube and aliquot. Freeze aliquots at -80°C. I suggest making 1mL aliquots. If desired, you can filter the virus containing media through a 0.2 micron filter prior to aliquoting.

Titer Lentivirus - 10/01/10

Day 1:

Plate 1 x 10⁶Hela cells in 35 mm dishes.

Day 2:

Infect with 100uL and 200uL lentivirus stock in a final volume of 200uL. Rock every 15 minutes for 1 hour. After 1 hour add 1mL of DMEM + 10% FBS + Pen/Strep to each plate. Do not remove the virus. Be sure to include 2 mock-infected controls.

Day 3:

Aspirate media and wash cells. Add complete media + 1-2ug/ml Puromycin to all infected dishes and one of the control plates. Do not add Puromycin to the second control plate.

Day 5:

Score cytopathic effect. All cells on the control plate treated with Puromycin should be dead and floating. Determine the least amount of virus needed to protect the entire monolayer. Consider this an MOI of 1. If the 100uL sample confers resistance to all the cells than the titer is 1 x 10⁷Transforming Units (TU)/mL.

Tips for packaging and use of HIV-based Lentiviruses:

This protocol was optimized with pLKO.1 but works well with all HIV-based lentiviral plasmids such as pLL5 and pLenti. This is a second-generation HIV-based packaging system. It will not work for pLPCX or pLNCX.

Lentiviral shRNA:

The following shRNA sequences have been cloned into the pLKO.1 expression system available from Addgene (http://www.addgene.org/tools/protocols/plko). I have optimized protocols for packaging lentivirus, titrating virus, and infecting target cells. I will attach my extended protocol, but a basic protocol is available in the above link and in the methods section of Mohni et al 2010 (1).

All of the plasmids are Amp resistant. I recommend growing the pLKO.1 based plasmids (or pLL5, pLenti) at 200µg/mL of Amp. Alternatively, I also use 150µg/mL of Carbenicillin. The pLKO.1 plasmids have repeat sequences and replicate rather slow, so you may also need to allow a little more time for your cultures to grow. You will need high quality DNA preps of at least 1mg/mL if you plan to transfect these plasmids directly or produce lentivirus particles with them, do not use Mini-Prep DNA.

I package VSV-G pseudotyped replication defective lentivirus particles using a second generation packaging system consisting of the packaging plasmids psPAX2 (expresses gag/pol) and pMD2.G (expresses VSV G). These plasmids are both Amp resistant and grow quite well. If VSV-G pseudotyping is not appropriate for you, I have several other ecotropic (murine only) and amphotropic envelope proteins for pseudotyping. I do not have any third generation packaging plasmids (which use separate vectors to express gag and pol).

Sequences for pLKO.1 inserts (The first red sequence is the specific targeting sequence and the second red sequence is its complement).