Tel Aviv University - Vector Core Facility
HEK293 cell culture
HEK293 cells are grown in full DMEM medium containing 10% FCS, L-Glutamine, pen/strep, sodium pyruvate and non-essential amino acids. Cultures are split 1:10 three times a week before they reach full confluence. Click here to view the full protocol.
Transfections of HEK293 cells
Two days prior to transfection, confluent cell cultures are split 1:10 and seeded in new 10cm plates containing full DMEM medium. On the day of transfection, the full medium is replaced with transfection medium, which contains 1-2% FCS.
Transfections of viral plasmids is carried out when the cultures reach 70%-80% confluence, either by using either calcium-phosphate precipitation, or polyethilene-imine (PEI): for each plate, 20µg of vector plasmid are used, along with its respective helper plasmids. The DNA transfection mix is added dropwise to the plates, and is replaced with full DMEM medium after eight hours. For letivirus and retrovirus transfections, 6ml fresh medium are added, and to AAV plates 10ml.
Virus colloection
Lentivirus
Supernatant from transfected cultures is collected twice; 36 and 48 hours post-transfection. After each collection, the supernatant is centrifuged at low speed and than filtered using a 0.45µm filter. This supernatant contains virus concentrations ranging between 10^5 - 10^7 TU/ml, depending on the size of the insert and can be used to infect cultured cells. If this is the desired end-product, HEPES solution at pH 7.2 will be added to a final concentration of 50mM and the supernatant will be haliquoted to ten tubes containing 500µl each and kept at -80⁰ till use.
AAV
At 72 hours post-transfection, cells from the plates are scraped, collected into 50ml tubes and centrifuged at low speed in order to pellet the virus-containing cells. The medium is then aspirated from the tubes and the cells are resuspended in a lysis buffer containing 150mM NaCl and 50mM Tris-HCL at pH 8. After resuspension the lysate is subjected to three rounds of freeze-thaw cycles between -80⁰ and 37⁰. Next, benzonase nuclease (Sigma #E1014) is added to the lysate (1hr in 37⁰), in order to digest the nucleic acids of the cells along with the transfected DNA. The lysate is then filtered through a 0.45µm filter to produce crude lysate suspension. This suspension contains virus concentration of ~10^8-10^9 TU/ml and can be used to infect cells in culture with high efficiency. If this is the desired end product, The lysate will be delivered in a single tube containing 1ml of lysate, kept at -80⁰. AAV are not sensitive to temperature changes and the lysate can be thawed each time prior to use or simply stored at 4⁰, where it can remain stable up to six months, if the use is frequent.
Virus purification and concentration
Lentivirus
Lentivirus or retrovirus concentration is accomplished using high-speed centrifugation. The supernatant from the first collection is distributed among 2-6 Beckman-Coulter ultraclear, 38.5ml centrifuge tubes. To the bottom of each tube, 4ml of 30% sucrose solution is gently pipetted in order to cushion the virus and keep smaller proteins from depositing in the pellet. The virus is subsequently centrifuged using an SW28 rotorhead at 25,000rpm for 1.5-2 hours at 10⁰. At the end of the first cycle the medium is aspirated from the tubes and the virus-containing pellets are resuspended in cold PBS. After the pellets from all tubes have been resuspended, the second virus collection is added and centrifuged in the same fashion as the first. At the end of the second cycle the pellets are again resuspended using the virus suspension collected in the first round. The suspension is then pooled, haliquoted in small PCR tubes containing 5µl each and kept at -80⁰ till use.
AAV
In order to separate the viruses, the crude lysate is passed through a column of heparin-agarose (Sigma #H6508), which binds with high affinity AAV capsid proteins of serotype 2. After addition of the lysate to the column it is kept at 4⁰ for 2 hours with constant agitation, at the end of which the flow-through is discarded and the virus can be eluted from the column using a high NaCl solution. Following elution, the virus suspension is desalinated and further concentrated using an Amicon Ultra Filter (Millipore #UFC9 100 24) to a final volume of ~150µl and then collected into a single tube and kept at -80⁰ till use.