Lentivirus infection

Vectors used (DOI: 10.7554/eLife.77444):

LCB = lentiCRISPR v2 blast (map attached) – source of Cas9, Addgene #83480

ipUSEPR (provided by Francisco Sanchez-Rivera, MIT) – source of guide

pEGPN - lentivral vector for overexpression

PAX2- packaging plasmid (gagpol) Addgene #35002

MD2G – packaging plasmid (VSVG envelope protein) Addgene # 12259

Day -1

1. Plate 5 million 293T’s per 10cm dish in P/S-free DMEM+10%FBS

Day 0

1. For one 10cm plate (scale by surface area for smaller wells): In one eppendorf tube per construct, add 500ul OPTI-MEM, plus 2.5ug lentiviral construct, 1.875ug PAX2, 0.625ug MD2G.
2. For each tube, add mastermix containing  500ul OPTI-MEM + 25ul Xtreme Gene9 (Roche/Sigma Aldrich 6365787001) for each construct.
3. Mix 7x by pipetting. Incubate 20 min RT.
4. Add dropwise to packaging cells

Day 1

1. Change media. (Safety note: put discarded media into bleach, use labcoat, double gloves when handling lentivirus).

Day 2

1. Carefully remove and store conditioned media, replace with fresh media.

Day 3

1. Collect media and combine with previous collection. To avoid contaminating target cells with 293Ts, clear cells + debris by filtering through 0.44um filter or centrifugation (2 rounds of 1500rpm x 10 min x 4 deg C). Bleach packaging cell plates.
2. Using freshly collected supernatant used immediately results in highest titers. However, lentivirus will be stable enough to use after a week stored at 4 deg C or long term if stored at -80 deg C. For a construct that can be used for multiple experiments (i.e. Cas9 with no guide), making a large batch and tittering once for each cell line is advisable.

Infection:

1. Add lentivirus to target cells at a dilution previously determined by titering virus (see optimization experiments below) or use a range of dilutions and determine fraction of infected cells by fluorescent expression or drug selection if applicable. Add ~4ug/ml final concentration polybrene (Santa Cruz Biotechnology, sc-134220).

Day 4 

1. Change media on target cells.

Day 5

1. Split target cells into selection. Ideally you will have previously performed a cell-line specific kill curve for the relevant selection marker. (Typical selection concentrations: 500 ug/ml G418/neo, 5 ug/ml blast, 2 ug/ml puro)

Day ~8-10

1. For CRISPR experiments, if cells look like infected at a reasonable titer, they will be ready to assess knockout efficiency or can be put into a functional assay 5-10 days after infection of the sgRNA.

Optimization experiments:

Selection. In each cell line used it will be helpful to have previously completed a dose-response experiment with any selection drugs to be used (G418/puro/blast). Test relative cell number after 3 days after treatment with 1:2 serial dilution centered around the typically used concentrations (see step 12).

Infection. Ideally any virus prep and cell line used, you will determine the dilution needed for a desired MOI (multiplicity of infection). Generally, infection with an excessively high copy number of construct will amplify both systemic and sequence-specific off-target effects. This is especially true with PLKO stem-loop shRNAs, for example. If nearly every cell is infected prior to selection, there will be a risk of artifactual findings.

For a construct that will be used for many experiments (Cas9 expression with no guide), it is convenient to make small aliquots and store them at -80 deg C. This will result in lower infectivity, but it will be consistent.

1. To titer virus, infect cells with a serial dilution of virus with a constant 4ug/ml polybrene. For example, in a 6W plate 1:4, 8, 16, 32, 64 and no infection.  Note that some cell lines might be sensitive to polybrene and concentration should be modified accordingly. 
2. For constructs that express a fluorescent marker, determine the % of cells infected by flow cytometry.
3. For constructs with a drug selection marker, split the cells +/- selection and use the ratio of cell number in the selected plate to the number of cells in the unselected plate to estimate the % of cells infected.

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