Transfection of HEK293T cells must be performed at 90%confluency. Also, the use of a 0.45-micron low protein binding filter for lentiviral vector filtration and the use of a sucrose cushion for ultracentrifugation are essential. Begin by culturing HEK293T cells in DMEM medium containing 10%FBS and 1X penicillin streptomycin at 37 degrees Celsius in a humidified incubator with an atmosphere of 5%carbon dioxide and 21%oxygen until cells reach 90%confluency before seeding for transfection.
Seed four times 10 to the six cells in 10 milliliters of complete growth medium in a 100-millimeter tissue culture plate and grow overnight in a humidified tissue culture incubator with an atmosphere of 5%carbon dioxide and 21%oxygen. For each transfection, dilute one milligram per milliliter of plasmid DNA stocks in one milliliter of serum-free DMEM media in 1.5-milliliter centrifuge tubes by following the ratio of entry and packaging plasmids as described in the text manuscript. Vortex the tube for 10 seconds and spin at 10, 000 times G for 30 seconds at room temperature to collect.
Then, add 70 microliters of one milligram per milliliter stock solution of polymer polyethyleneimine, or PEI, to the tube. Again, vortex and briefly spin the tube. For lipofectamine-based transection, dilute the vectors in 500 microliters of serum-free DMEM media containing 45 microliters of reagent P supplied in the kit.
Then, dilute 70 microliters of reagent L using 500 microliters of the same medium. Incubate both the tubes at room temperature for five minutes. Then, combine the contents of both tubes and incubate at room temperature for 20 minutes to allow complex formation.
Next, add drop-wise one milliliter of DNA PEI or DNA lipofectamine complexes to the plate containing cells and incubate for six hours at 37 degrees Celsius with an atmosphere of 5%carbon dioxide and 21%oxygen. After incubation, replace the media with 10 milliliters of fresh complete growth media and return the plate to the incubator. After two days of transfection, collect the virus-containing media and overlay the cells with 10 milliliters of fresh complete growth media.
Again, after three days of transfection, collect the virus-containing media and combine it with the media collected at the two-day time point. Centrifuge the viral supernatant at 2, 000 times G for 10 minutes at four degree Celsius to pellet cellular debris. Filter the supernatant using a 0.45-micron pore size low protein binding filter and store at four degrees Celsius until ready for ultracentrifugation.
Sterilize the ultracentrifuge tubes'holding cups by washing with 70%ethanol for 10 minutes. Air dry, close, and place the cups at four degree Celsius. Add 36 milliliters at filtered media containing lentiviral particles to a sterile ultracentrifuge tube.
Fill five milliliters of a sterile stripette with four milliliters of sterile 20%sucrose solution prepared in PBS and dispense it to the bottom of the tube containing lentiviral vectors, or LVS. Ensure that the sucrose solution should not be mixed with the media and makes a gradient at the bottom of the tube. Balance all the ultracentrifuge tubes using serum-free DMEM media, place the tubes in cold ultracentrifuge tube holding cups, and close the lids.
Spin the tubes at 125, 000 times G for two hours at four degrees Celsius. After the spin, carefully discard the supernatant by inverting the contents of the tube in a container containing bleach without disturbing the pellet. Mark the pellet if visible.
Place the ultracentrifuge tube in a 50-milliliter sterile tube and add 200 microliters of sterile DPBS at the top of the pellet. Place the tube at four degrees Celsius overnight. The following day, gently mix the content of a tube by pipetting up and down before spinning at 13, 000 times G in a tabletop centrifuge to pellet any debris.
Transfer the supernatant to a new tube and aliquot the virus preparation. Finally, store the tubes at minus 80 degrees Celsius. After transfection, abundant GFP expression was observed in HEK293T cells.
The expression of viral proteins resulted in syncytia formation of HEK293T cells where single cells are fused. Following infection, marked expression of GFP was also observed in viral-infected neural progenitor cells. LVS titer measurement between the low-cost PEI and lipofectamine 3000 reagent did not show any significant difference in viral titer.
The viral titers for PLK 0.1-based vectors are shown here. Lentiviral transfected cells showed more than 80%cell viability as compared to non-transduced cells, while no cells survived the treatment. Western blot analysis showed abundant expression of L-2-hydroxyglutarate dehydrogenase in cells transduced with an empty vector backbone.
However, no expression was observed in cells transduced with a vector carrying short hairpin RNAs. The main advantage of this technique is the use of low-cost cationic polymer polyethyleneimine to produce lentiviral particles by co-transfecting HEK293T cells using entry and packaging vectors.
