Note: 2x 10cm plates (when concentrated down to 100 µl), should give titres ... Cover falcon tube containing filtered virus supernatant with parafilm & place into ...
PRODUCTION & PURIFICATION OF LENTIVIRUS (Small scale, 2nd Generation): REAGENTS: - Lentivirus vector [enter customer details] - Lentivirus packaging vectors (pMD2.g & psPAX.2) - p24 Elisa Kit (NEN Life Science; Cat #: NEK050B) – optional* - 1x PBS - 1x HBSS * Viral titre can also be determined by FACS (if there’s a GFP tag) or using the p24 ELISA kit in accordance with manufacturer’s instructions found on the following website: http://las.perkinelmer.com/Content/manuals/man_hiv1p24elisa.pdf EQUIPMENT: - 10 cm tissue culture dishes - 1 plate per vector (if customer ordered viral supernatants only) - 2 plates per vector (if customer ordered 100 µl of concentrated virus) - 0.45 µm filter units, 500 ml capacity (Corning; Cat #: 430773) - Round bottom ultracentrifugation tubes (Beckman; Cat #: 326819) - Tissue culture incubator at 37ºC/5% CO2 - Beckman SW41 rotor (or equivalent) - Beckman ultracentrifuge (or equivalent) - Fluorescent microscope - Multichannel pipettor - Microplate reader - Fluorescence-aided cell sorting (FACS) capabilities CELL LINES: 293T HEK cells (If possible, use 293FT cells as they give you a higher titre) PROTOCOL: Day 1) Prepare Reagents: - CaCl2 stock solution (2.5M CaCl2): - 36.75 g of CaCl2 in 70 ml ddH2O - Take to final volume of 100 ml, aliquot into 1.5-ml eppendorffs - store at –20°C - 2x HBSS solution - 274 mM NaCl (Baker; Cat #: 3642-05) - 10 mM KCl (Sigma) - 1.4 mM Na2HPO4 (Baker) - Add ddH2O up to 900 ml. - Dissolve & titrate to pH 7.05-7.11 with 1M NaOH Note: pH must be within this range, as even a slight increase/decrease in your pH will reduce transfection efficiency. - Bring volume to 1 L - Filter sterilize, aliquot 1 ml into 1.5 ml eppendorf tubes & store at -20 ºC (long term storage) or at 4ºC. Note: once thawed, do not re-freeze
Low passage, 293T cell preparations: - Harvest cells from 2 x10 cm plates of 293T HEK cells grown in DMEM + 10% FBS. - Cells must be low passage & do not allow them to be over-confluent (max 60%) (Note that if you allow these cells to overgrow at any stage (beyond 70%), your transfection efficiency will be reduced significantly) - Split 293FT cells amongst the 10 cm plates (750,000 cells per plate) using DMEM + 10% FBS medium - Swirl the plate when seeding to ensure cells are spread evenly across the plate surface - Final volume of media = 10 ml - Incubate plates at 37ºC, overnight Day 2) Transfection with plasmid mix using CaPO4 * Check cells are ready for transfections - They should be around 35-40% confluent Note: do not use cells if they are overgrown as it will significantly reduce transfection efficiency *Prepare your “virus plasmid mix + reagents” in an eppendorf tube as follows (IN THIS ORDER): -
Add ‘x’ µl of ddH20 to the tube Add 17.5 µg of the “customer’s vector” Add 5 µg of psPAX.2 Add 2.5 µg of pMD2.g Add 50 µl of 2.5 M CaCl2
Total volume = 500 µl
* Add 500 µl of 2x HBSS to each tube dropwise - Cap the eppendorf tube & invert 5-6 times after every 3-5 drops * When all of the 2x HBSS has been added, mix the tube by gently inverting the tube 5-6 times * Incubate at RT (22–26°C) for 15 min * Add the transfection mixture to each plate - spread the mixture around the plate drop-by-drop * Swirl plates gently * Incubate overnight at 37°C/5% CO2 (approx 16-20 hr) Note: 2x 10cm plates (when concentrated down to 100 µl), should give titres around 109-1010 Day 3) Check transfections: - Cells should be reaching full confluency - Check fluorescence (if plasmid has a fluorescent tag) - Transfection efficiency should be around 90% Note: when you convert from “bright light” to “fluorescence” you should be able to see that approximately 90% of cells are positive for fluorescence. - Remove media - Add 10 ml of fresh DMEM + 2% FBS to each dish - Incubate overnight at 37°C/5% CO2
Day 4) Viral Harvest # 1: - Collect & pool supernatants (if you have multiple plates per vector) - Spin at 1500 rpm for 5 min to pellet any cells/cellular debris - Collect supernantant with a syringe & filter through a 0.45 µm syringe filter into a “NEW” pre-labelled falcon tube - Cover falcon tube containing filtered virus supernatant with parafilm & place into sealable bag (double containment) - Store at 4ºC overnight in a cardboard box - Add 10 ml of fresh DMEM + 2% FBS to each dish - Transfer to 37°C/10% CO2 incubator - Incubate overnight Day 5) Viral Harvest # 2: - Collect & pool supernatants (if you have multiple plates per vector) - Spin at 1500 rpm for 5 min to pellet any cells/cellular debris - Collect supernantant with a syringe - Pass supernatant through a 0.45 µm syringe filter into the falcon tube containing your filtered “Viral Harvest # 1” supernatant from the previous day Note: The cleared supernatant may be used to directly transduce cells & titre will be relatively low (~106 viral particles/ml) Day 5) Virus concentration (for in vitro grade stock): - Centrifuge the supernatant using conical tubes and a Beckman ultracentrifuge with a SW41 swing bucket rotor (makes it easier to detect the viral pellet) - spin at 24,000 rpm for 1 hr at 4°C - carefully poor off supernatant without disrupting viral pellet - add last remaining supernatant & spin again (do not use these tubes more than 3 times) - carefully poor off supernatant - allow remaining supernatant to drain off by resting tubes on paper towel - aspirate all remaining fluid - Resuspend viral pellet in 100 µl of 1x HBSS (no frothing) - Rinse tubes with 50 µl of 1x HBSS & pool the samples (total volume per tube = 150 µl) - Transfer to ‘screw-top’ microcentrifuge tubes or eppendorf tubes covered with parafilm - Vortex at low speed for 15-30 min - Spin 10 seconds using a table top microcentrifuge - Collect supernatant (140 µl). Note: Use 40 µl (max) to determine viral titres by FACS or p24 ELISA. Customers get 100 µl Short term storage (< 1 mth) at -20°C; Long term storage (> 1 mth) & at -70°C.
