This procedure begins with transecting 2 9 3 T cells with lentivirus, plasmid and viral packaging vectors, the viral supernatant is harvested after at least a 48 hour incubation period. The snat is filtered to be concentrated with an ultracentrifuge after ultracentrifugation. The viral pellet is resuspended with PBS to obtain virus titer, first dilute concentrated virus in complete media, then perform serial dilution to infect cells with lentivirus.
Hi, I'm Shein Wong from the laboratory of Michael McManus at the Diabetes Center in the University of California San Francisco. I'm Michael McManus. Today we'll show you a procedure for the production of lentiviruses.
We use this procedure in the laboratory to make lentiviruses for RNA interference. So let's get started. To begin this protocol, prepare the DNA with which to transfect the 2 9 3 T cells.
First dilute FU gene six transfection reagent in a 1.5 milliliter tube by mixing 30 microliters Fuji six with 600 microliters serum free medium. Be careful to not let Fuji touch the side of the tube as you deliver it into the medium incubate room Temperature for five minutes. Next, in the cap of the tube, mix four micrograms lentiviral plasmid with 1.33 micrograms.
Each third generation viral packaging vectors. The lentiviral plasmid contains the DNA. The virus will insert into the genome of every cell infects while the packaging vectors contain genes for all the other proteins required to make a lentil virus close the lid and mix by inverting the tube a few times.
Incubate the DNA and transfection reagent for 15 to 20 minutes at room temperature. Now it is time to transfect 2 9 3 cells with the DNA you have prepared use cells that you have plated 24 hours in advance and that have reached 50 to 70%co fluency in the tissue culture hood pipette all of the prepared DNA onto the 2 9 3 plate mixed gently by tilting the plate back and forth, return the cells to the incubator and allow viral production to continue for 48 to 96 hours before harvest. During incubation, the 2 9 3 cells produce lentiviruses whose genomes contain an RNA copy of your DNA of interest.
Now it is time to collect the virus. Take your cells out of the incubator. Have ready in the tissue culture hood, a 0.4 or five micron syringe filter, a disposable 10 milliliter syringe and an UltraClear Beckman centrifuge tube.
Use the disposable syringe to remove the supernatant, which now contains the virus. Attach a 0.45 micron syringe filter to the tip and filter the virus into an UltraClear Beckman centrifuge tube. The filter allows only viruses and not cells to pass through before discarding, incubate all virus producing cells.
Culture plates, syringes and filters in 10%bleach for 45 minutes. Now that you have collected and filtered the virus, it is time to concentrate the virus by ultracentrifugation first. Use serum free DMEM to balance all virus containing tubes to within 0.2 grams of each other.
Next, load the balanced centrifuge tubes into SW 41 TI or SW 28 rotor buckets. Seal the rotor buckets shut and hook them onto the rotor. Then place the rotor inside the ultracentrifuge.
Spin the virus for about 90 minutes at four degrees Celsius at 25, 000 RPM in an SW 41 TI rotor, or 24, 000 RPM in an SW 28 rotor. After centrifugation, return to the tissue culture hood and invert the tube to pour the SNAT into a container containing 10%bleach. Use a Kim wipe to absorb excess liquid around the pellet and invert the tube in a convenient rack for no more than five minutes until you are ready to resuspend the pellet.
To resuspend the viral pellet, use a filter tip to add 100 microliters of sterile pre-filtered PBS to your tube. Then pipette up and down about 20 times. Leave the virus at four degrees overnight to complete resuspension.
You can store viral preps at four degrees for about one week and at minus 80 degrees for up to one year. Remember to treat all empty tubes with 10%bleach before discarding. If you intend to use the viral prep for longer than the one week, it will keep in the fridge.
Make minimal aliquots of five to 20 microliters for future experiments and one aliquot of three to five microliters for the titration freeze and store the aliquots at approximately minus 80. The next step is to determine the viral titer. If you have included a fluorescent reporter gene in your lentiviral plasmid infected cells will fluoresce and you can determine the viral titer using flow cytometry.
To begin, dilute three microliters of concentrated virus into 1.5 milliliters of complete medium in wells, one to six. In a single row of a 96 well plate. Perform the following serial dilutions to all six wells at 100 microliters of complete DM.To the first well add 100 microliters of medium with no virus.
This is your unstained control. To the second, well add 100 microliters of the diluted virus to the third. Well add 100 microliters of the mix from the second well to the fourth.
Well add 100 microliters from the third well to the fifth. Well add 100 microliters from the fourth well and to the sixth. Well add 100 microliters from the fifth.
Well finally take 100 microliters out of the sixth well and discard and bleach bring the total volume in all of the wells to 200 microliters with DMEM. With each dilution, the viral concentration decreases by half. Note that these are only suggested dilutions, which should work well for titering most concentrated viruses.
If your virus is low in titer or unconcentrated, you may need to adjust your dilutions. Treat any unused diluted virus with 10%bleach for 45 minutes before discarding into a biohazard waste container. Add about 15, 000 healthy actively dividing 2 9 3 T cells to each of the six wells.
You can use a single 96 well plate to titer 16 viral preps, top off wells to 200 microliters, return cells to the incubator and allow infection to proceed for at least 48 hours. After the incubation, analyze the cell fluorescence using a flow cytometer. You can use the percentage of fluorescent cells per well to determine the titer or number of infectious viral particles per milliliter of virus added.
Calculate the number of infectious units per milliliter with the following formula when interpreting flow. Cytometer results. Note that one can only achieve accurate titer calculations when infected cells have received no more than one viral integration per cell.
In order to ensure that this is the case, use cells with less than 15%infection rate for calculations. Cells with higher infection rates will likely have received multiple viral integr per cell. The dilution suggested in step two should yield several samples within this range of infection.
Ideally use several samples to generate a titration plot from which you can calculate the final titer. The calculated formula from the fitted linear line can be used to calculate the viral titer, but you can calculate titer using a single sample if necessary. One can expect titer of one times 10 to the seventh transducing units per milliliter for unconcentrated virus, and one times 10 to the eighth transducing units per milliliter for concentrated virus.
We've just shown you how to produce lentiviruses when doing this procedure. It's important to adhere to all biosafety regulations and dispose of waste properly. So that's it.
Thanks for watching and good luck with your experiments.
