From 2007.igem.org

Making a Lentivirus

We used 293 FT Cells (Human Embryonic Kidney cells). Get them from the -80 C freezer (short term storage) or liquid nitrogen (long term storage). 1. Warm cells in a 37 C bath, thaw until only a tiny chunk of frozen material is left. 2. Add cells to 5 mL of medium. 3. Spin down cells once you put them in the 5 mL medium for 5 minutes at 300 rcf. 4. From spun down cells using Pasteur pipette suck out supernatant, add 700 ul of medium, resuspend. 5. Spread over a 15 cm dish with 25 mL medium or 10 cm dish with 10 mL medium and swirl carefully.

• Incubator = 37 C and 5% CO2 and water at bottom. • Anything going into hood gets sprayed with 70% EtOH

Culture/Growth Medium: 0.85 liter of DMEM (from Invitrogen) 100 mL of serum (all purpose fetal calf serum; not ES cell qualified serum!) 10 mL of 100mM sodium pyruvate (from GIBCO) 10 mL of 100x MEM NEAA (non-essential amino acids from GIBCO) 10 mL of 200mM L-glutamine (from GIBCO) 10 mL of 100x 2-Mercaptoethanol (From SIGMA) (To make 100x stock, add 37 uL of Mercapto Ethanol = 14.2 M, to 100 mL of PBS) 10 mL of Penicillin-Streptomycin 100X (from GIBCO)

Splitting Cells

1:4 split (or 1:5 depending on cell density)

1. 25 mL medium for each of 4 (or 5) 15cm dishes 2. Remove medium from first dish (dish with cells) with Pasteur pipette 3. Add 2ml of Trypsin (brand: TrypLE Express) to first dish, swirl around and try to get all the cells off 4. Add 2ml medium to first dish and pipette up and down to get the remaining cells stuck on the plate off 5. Distribute all 4 mL between the 4 (or 5) new dishes. 6. Wait three days (or until confluent) to harvest

Day -1: Preparing for transfection

1. Carefully suck up medium from plates 2. Add 2ml of trypsin to each plate, distribute it evenly on each plate 3. add more medium to each plate, like in cell splitting (medium inactivates trypsin) 4. Transfer medium and cells from plate to a tube (50 ml tube). Make sure to wash all of the cells off the plate. Use as much medium as necessary but don’t make it too dilute. It should look transparent, not cloudy.

Counting

1. Mix tube gently by inverting up and down. 2. pipette 10 ul onto a microscope slide but don’t overfill slide 3. Count cells under microscope using a colony counter. 4. The slide is a Reichert hemocytometer with 4x4 corners and 5x5 middle. All large squares have the same area. 5. Cells/ml = # cells counted * dilution factor * 10^4

Count the number of cells per large square (4x4 or 5x5). They all have the same area. For cells that overlap between squares, count only those on two sides (eg. left and top).

Day -1: Plating 1. Coat 3 plates/plasmid made with 0.1% gelatin in Ultra Pure water (15-20 ml). Cover for at least an hour at room temperature. 2. Remove the liquid, the gelatin should stick to the plate. 3. Add ~20 ml of medium to the plates 4. You want to plate 1.8-2.0 * 10^7 cells per 15 cm dish 5. With extra cells that are left in the tube, replate to continue growth on a clean plate, also add ~25 ml medium to the plates

Day 0: Transfection 1. We have three 15 cm dishes per transducing plasmid. 2. Remove the medium from the plates and add carefully and slowly 12.5 ml of new growth/culture medium to each plate. Tip the plate and add the new medium gently to the side of the plate. You do not want to disturb the cells growing on the plate. 3. For each 15 cm plate, use: a. 18.9 ug of p148, b. 9.5 ug of p149 c. 7.43 ug of transducing vector d. and enough DMEM to make a total volume of 1110 ul

Add the DMEM First! Mix well and add 75 ul of SuperFect Transfection Reagent 4. Mix gently and incubate for 10 minutes at room temperature 5. Add the mixture dropwise and spread evenly across the plate. After all the medium is added, give it a VERY gentle swirl.

Day 1: If possible, move dish from 37 C / 5%CO2 to 32 C / 5%CO2. This step will extend the half-life of the viral particles and, hence, increase the titer.

Day 2: Viral Harvesting 1. Filter the supernatant from the transfected cells. Put 3 plates worth of cells in a “low protein binding membrane filter” (Corning 430625). 2. Put liquid in polycarbonate centrifuge bottles, balance them perfectly by adding medium (DMEM). 3. Spin at 50,000 g for 2.5 hours at 4 C, accelerate at max. Leave break off so as not to disturb the pellet of virus. It will take about 30-40 minutes for the centrifuge to stop spinning.

Storage 1. Carefully suck out medium without disturbing viral pellet. Tilt tube with pellet facing the ground, suck from the top. When liquid is past the level of the pellet tilt more and try to suck out all of the medium. 2. Add about 80 uL PBS (Phosphate Buffered Saline), resuspend by flicking with finger. If you see virus particles floating around, pipette up and down. 3. Put in 4 C fridge for 30 min 4. Divide into 5 tubes or more depending on pellet size, label and date. 5. Try to get everything into tubes ~20 ul per. 6. Add bleach to centrifuge tubes, also bleach all tips – bleach everything in contact with virus. Let stay at least 20 minutes. Then wash the tubes out with water followed by 80% ethanol (“sterilization”), and sterile water or PBS. Dry tubes.