GUgene_protoocol

Retrovirus Production – Fugene Protocolby Catherine KingJuly 2010Method by Greg Towerso These procedures should be performed in Level 2 containment facilities. Please contact your safety officer prior to beginning this work to ensure all safetymeasures and appropriate risk assessments have been put into place.o Please note that you will require different gag-pol packaging plasmids depending on the library vectors you are using. For GIPZ plasmids, you will require the gag-pol expressor p8.91 and for the pRetroSuperCam plasmids you will require the gag-pol expressor pMLV-GP. The same VSV-g envelope plasmid, pMDG, is used for both library plasmids.Aim To produce a retroviral stockTime scaleDay 1 Seed HEK293T cellsDay 2Co-transfect with hairpin construct (GIPZ or pRetroSuperCam) and retroviral packaging vectorsDay 3Change media on transfected cellsDay 4/5/6 Virus collectionMaintaining HEK293T CellsMaintain HEK293T cells in complete DMEM-10 or 15 (DMEM, 10 or 15% FBS). They should be split 3 times a week at a ratio of 1:4 - 1:6 using trypsin EDTA (i.e. Monday, Wednesday and Friday) Frequent passages and keeping the 293T as individual cells will keep the transfection efficiency high. Please also see notes below on happy 293T cells.Day 1 – Seeding HEK293T Cells•Seed HEK293T cells by splitting a confluent plate 1/4. Cells should be seeded approximately 24 hours before transfection into a 10cm plate with 8ml completemedia and incubated at 37°C, 5-10% CO2. Please note that cells should be sub-confluent at the time of transfection (see pictures in notes below). To reduce the scale1 million 293T/well in a 6 well plate works well (divide the rest of the protocol by 3 i.e.3ul fugene, 300ng p8.91 etc).Day 2 – Transfection•Dilute DNA in a 1.5ml eppendorf tube to a total volume of 15ul with TE pH8.0.1ug p8.91 or pMLV-GP (gag-pol expressor) *1ug pMDG (VSV-G expressor)1.5ug pGIPZ or pRSCam DNA *sterile TE to 15ultotal 15ul* for the GIPZ vectors you should use the gag-pol expressor p8.91 and forthe pRetroSuperCam vectors you should use the gag-pol expression vectorpMLV-GP•For each transfection mix, add 200ul Optimem to a second eppendorf tube and add 10ul Fugene to the centre of the tube without touching the sides of the tube. Flick tomix, do not pipette mix.•Add DNA to the Optimem Fugene mix and once again flick to mix•Spin for 2 seconds to remove the mix from the sides of the tube•Leave at room temperature for 15minutes.•During this incubation, change the media on the cells with fresh complete media that has been pre-warmed.•After the 15-minute incubation, add the Optimem/DNA/Fugene mix to the cells dropwise and swirl the plates to mix well.•Incubate cultures overnight at 37°C, 5-10% CO2. (Best to transfect mid afternoon.) Day 3 – Media Change•Approximately 24 hours following the transfection, replace the medium containing the transfection mix with 8ml of fresh complete medium.Day 4/ Day 5/ Day 6 – Virus Collection•Harvest retrovirus-containing supernatant 24, 48 and 72 hours after media change.•Collect medium into a 10ml or 50ml syringe and filter through a 0.22µm filter into a falcon tube.•Replace fresh complete media on the cells if you wish to collect the following day (same volume as on Day 3).•Supernatent can be stored at 4°C overnight for later concentration or stored in un-concentrated aliquots at –80°C. On a good day the titre should not reduce between day 4 and 5 and will be a little lower on day 6.Reagents required:To be obtained from the RNAi facility•p8.91 (gag-pol expressor for the GIPZ library)•pMLV-GP (gag-pol expressor for the pRetroSuperCam library)•pMDG (VSV-G expressor)•Individual pGIPZ and pRetroSuperCam clones from the libraryIndividual clones and packaging vectors will be provided as a bacterial stock. You should grow these bacteria according to the pGIPZ manual, that is in 100ug/ml Carbenicillin, and prepare the plasmid DNA using a Qiagen Maxi kit (12163) to produce high quality DNA for transfection.Other reagents•HEK293T cells (ATCC 293T/17 cell line. O rder from LGC in the UK # CRL-11268) •DMEM High Glucose 41966-052 Invitrogen •Qualified Fetal Bovine Serum- heat inactivated S1900 Biosera •Optimem with glutamax1 51985-026 Invitrogen •Fugene 6 transfection reagent 1988387/1815075 Roche Diagnostics•10ml syringe disposable SZR-150-052C Fisher•50ml syringe disposable SZR-150-071V Fisher•0.22uM Nalgene Syringe filters 513-1901 VWR•Ultracentrifuge tubes Polyallomer Konical 30ml 358126 Beckman CoulterEXTRA NOTES FROM GREG:Any questions call Greg on the 293T helpline 020 7679 9535 or g.towers@We can show visitors how we do all our retrovirus experiments by appointment.Good luckHappy 293T cellsThe titre of the virus absolutely depends on how you keep the 293T cells. Titres can vary between 105 and 108 i.u./ml but will be high if you keep the cells appropriately. The key is to keep them pretty dense most of the time. 293T come off the plate easily with trypsin but make sure you trypsinise them properly so that they are individual cells when you plate them. A few times up and down in a 10cm pipette helps. Remove bubbles and make sure the shelf in the incubator is level. We often let the cells stick down before we stack the plates. We split ¼ Mondays and Wednesdays and 1/6 on Friday. Use DMEM with Glutamax and high Glucose throughout. They must also be almost confluent when you transfect them. The aim is to transfect them at or just before the point they become confluent and then they don’t divide and virus release stays high for several days. If cells look ill during normal passage try 15% serum. Happy293T=very high virus titre. When you transfect them they should look like the first picture. You might be surprised how fast they grow. If they don’t look good in the morning leave them until last thing to transfect. The second is not confluent enough. The third picture shows what the cells look like when they’re not very well. They make a kind of reticular pattern and don’t plate evenly, note the gaps. They do this when they don’t like the serum, or when you’ve split them too hard. We batch test our serum using 293T.Concentrating virus.Spin 30ml of virus in a swing out rotor at 17K for 2 hours at 4 degrees. Pour off the media and resuspend in 1ml media without FCS taking care not to foam. Add FCS later if necessary. Resuspend with at least 40 pumps. You can spin through a sucrose cushion (5ml 25% sucrose in the bottom of the tube) but we tend not to as the sucrose is sometimes worse than the crap you’re trying to remove.When infecting cells with concentrated virus it’s often best to infect the cells first thing and then change the media last thing to reduce toxicity, i.e. don’t leave on the cells overnight. InfectionsWe infect cells plated at 105/well in 6 well plates in 1ml in the presence of 8µg/ml polybrene. Make polybrene at 1000X in water and filter (0.45µm).You can increase the titre by spinning the virus onto the cells. We use 500G (1.6K in a Sorvall Legend) for 1 hour at room temp in a bench top centrifuge. Use 1.5ml/well in a 6 well in this case. After spinning leave the sup on the cells overnight and then change it. If you have toxicity reduce the time the virus is on the cells but don’t go below about 4 hours if you can help it。