Lentivirus Production

Transcription

1 Lentivirus Production Day 1 (morning): Plate 2.5X LT cells/10cm dish. It is critical that the cells are properly maintained for optimal virus production: Cells must never grow more than 80% confluent; cells should be lower than passage 20. Plate 400,000 cells for 6-well or 35 mm plate Plate 6.5X10 6 cells for a 15 cm plate. Day 2 (morning): Prepare PolyJet transfection as follows for each 10cm plate*: 1. For each 10 cm dish, replace medium with 5.0 ml of fresh complete medium with serum and antibiotics 30~60 minutes before transfection. 2. Dilute in 500 ul high glucose serum-free DMEM: i. Lentivector 8.3 ug ii. pspax2 4.2 ug iii. pmd2.g 2.5 ug iv. Vortex for 5 sec. 3. Dilute in 500 ul high glucose serum-free DMEM: i. 45 ul PolyJet 4. Vortex for 5 sec. 5. Add diluted PolyJet to diluted DNA (IMPORTANT TO ADD POLYJET TO DNA, NOT DNA TO POLYJET). 6. Vortex for 5 sec and incubate at room temp for 15 minutes. All subsequent steps must be performed in the lentiviral TC room. 7. Add DNA/PolyJet dropwise to 293 LT cells. 8. Incubate cells at 37 o C for 4 hrs. PolyJet is toxic to cells and the cell viability will be reduced if PolyJet is left on the cells for more than 4 hrs. 9. Aspirate transfection reagent and add 8 ml of fresh growth medium (DMEM + Pen/Strep + 10% FBS). 10. Return to TC incubator and incubate for 48 hours at 37 o C to produce virus.

2 For 6-well or 35 mm plate, divide everything by 6. For 150 mm plates, multiply everything by 2.5. Day 4 (anytime): 1. Collect supernatant in a 15 ml tube. A 50 ml tube can be used if more than 15 ml of viral supernatant is being collected. 2. Centrifuge at 2500 rpm for 5 minutes. 3. Transfer the supernatant to a 50 ml conical tube using a 10 ml serological pipette. Note the volume transferred. 4. Add a volume of 40% PEG that is equal to 1/3 the volume of the viral supernatant. For example, if you transfer 9 ml of viral supernatant, add 3 ml of 40% PEG. The PEG is very viscous and you will need to gently pipette the viral supernatant up and down to rinse the PEG out of the pipette. CAUTION make sure not to blow air out the pipette, as this may cause viral aerosols. 5. Cap the 50 ml tube and invert 5-6 times to completely mix the viral supernatant and PEG. 6. Incubate at 4 o C overnight. The virus precipitate will have a white fluffy appearance. 7. Centrifuge the precipitated virus at 4000 x g for 30 min at 4 o C. 8. Aspirate off the supernatant. A small white pellet will be visible at the bottom of the 50 ml tube. 9. Resuspend the viral pellet with 250 ul of culture medium using a P1000 pipette. Use MEC medium or the medium that will be used to culture the cells in. Take extra care not to touch the barrel of the pipette to the wall of the tube, as this will contaminate the pipette. 10. Transfer the virus concentrate to a 1.5 ml screw-top tube. 11. Transfer 5.5 ul of viral concentrate to a 1.5 ml screw-top tube. Label the tube with the name of the virus, date and an asterisk. This will be used for testing the virus titer. 12. Aliquot the rest of the viral concentrate (50 ul/tube) into 1.5 ml screw-top tubes. 13. Store virus at -80 C.

3 Virus is stable for at least 6 months. Thawing the virus will decrease the titer. The titer is approximately halved for each thaw. Titering Lentivirus 1. In each of four 1.5 ml snap-top tubes add 20, 000 cells in 500 ul total volume. Trypsinize 293LT cells by aspirating medium, adding 1 ml trypsin/edta and incubating in the TC hood (RT) until the cells detach from the plate and each other. Add DMEM with serum to inhibit trypsin and trasfer cells to a 15 ml tube. Centrifuge at 800 rpm for 3 minutes to collect cells. Aspirate medium and resuspend in 5 ml DMEM + Pen/Strep + 10% serum. Count cells with haemocytometer. Calculate the total number of cells you will need for tittering (i.e. you will need 4 X 20,000 for each virus being tittered.) Transfer the required number of cells to a 15 ml tube and add complete DMEM (DMEM+Pen/Strep+10%FBS) to the total volume required (i.e. for each virus, 4 X 500 ul = 2 ml). (e.g., For tittering 2 viruses, 20,000 cells X 8 = 160,000 cells. 8 X 500 ul = 4 ml. You will need 160,000 cells in a total of 4 ml. Using a P1000, transfer 500 ul to each tube. 2. Thaw the 5.5 ul virus aliquot and add it to tube #1. 3. Make 1/10 serial dilutions to the other three tubes. With a P200 set to 50 ul pipette, gently up-and-down 10 times to mix the virus and cells. Transfer 50 ul from the 1st tube to the 2nd tube. Mix up-and-down 10 Transfer 50 ul from the 2nd tube to the 3rd tube. Mix up-and-down 10 Transfer 50 ul from the 3rd tube to the 4th tube. Mix up-and-down 10 Transfer 50 ul from the 4th tube to the 1st tube. Mix up-and-down 10 This makes it so all tubes still have 20,000 cells. This results in 4 different amounts of virus in each tube, 5 ul, 0.5 ul, 0.05 ul, ul. 4. Transfer the cells with virus to a 24-well plate. Start with the cells containing the most dilute virus and transfer it to a well. Make sure to touch the tip of the pipette to the side of the well when pushing out liquid. CAUTION do not blow out by pushing the pipette plunger all the way down as this can generate lentiviral aerosols. If you work by transferring the most dilute to the most concentrated virus, you can use the same tip throughout. 5. Place plate in the lentiviral TC incubator and incubate for 72 hrs at 37 o C.

4 6. Fix in 4% PFA for 20 minutes. Add 100 ul of 20% PFA directly to cells containing ~500 ul of growth medium. Do not aspirate off the medium first, or else the cells will come off the plate. After fixation is complete, spray the outside of the plate with Conflikt The remaining steps can be completed at the lab bench. 7. Aspirate and rinse with PBS. 8. Stain nuclei by adding Hoechst (or 33258) diluted 1:5000 in PBS. 9. Incubate for 15 min at room temp. 10. Aspirate and rinse with PBS. 11. Take images at 32X using fluorescent microscope. Pairs of images are necessary nuclei and GFP. First, quickly scan each of the 4 dilutions to determine which has 1-10% GFP-positive cells. For an accurate calculation of the virus titer, cells need to have between 1-10% GFP-positive cells. If it is less than 1% then it will not be accurate because some fields will not have any GFP-positive cells. If it is greater than 10%, the likelihood of multiple integrations is increased and the titer will be underestimated. After finding the well with 1-10% GFP-positive cells, select a field with approximately cells (this is even coverage of cells in the field of view, but the nuclei of cells are easily distinguishable and separated. Take an image of the nuclei (blue channel) channel, then switch to the green channel and take an image of GFP-positive cells. Save the images, and analyze using ImageJ. The LV titer macro can be used to rapidly count nuclei automatically. Take 3 sets of images for each virus. Calculate titer: 1. Calculate the % of GFP positive cells for each image set: GFP/Nuclei = % GFP-positive 2. Average the % GFP-positive cells for the 3 image sets. 3. Calculate the total number of viral transducing units (TU): Avg. %GFP X # of cells plated (i.e ) = # transducing units (TU) 4. Correct for the dilution of the virus: TU/virus dilution (i.e. 5, 0.5, 0.05, ul) = TU/ul 5. Correct so that the titer is expressed as TU/ml TU/ul X 1000 ul/ml = TU/ml Typical values are for lentivirus with shrna. Typical values are for lentivirus with cdna.

5 Example: 1. You determine that from the 3 rd dilution (i.e ul virus) the following numbers are counted Nuclei GFP % GFP (7.2%) (7.2%) (6.9%) 2. Average GFP-positive = (7.1%) 3. TU = X 20,000 cells. = 1,420 TU 4. TU/ul = 1,420 TU/ 0.05 ul = 28,400 TU/ul 5. TU/ml = 28,400 TU/ul X 1000 ul/ml = 2.84 x 10 7 TU/ml