|Harvesting recombinant baculovirus|
|Initial (P0) recombinant baculovirus stock. 60-72 h after co-transfection (total incubation time) collect 1.5 ml of the transfection cell culture medium into an Eppendorf tube. Centrifuge in a microfuge for 5 min at 2000 r.p.m. to remove the cell debris. Collect the supernatant and store this initial, passage zero (P0) virus stock at 4oC. The initial virus stock can be used for a preliminary protein expression study although use of an amplified stock is preferable. Immediately use the remainder of the transfection medium (0.5 ml) for the generation of a high-titer P1 baculovirus stock (see below).|
|High titer baculovirus stocks|
Generation of high titer (P1) recombinant baculovirus stock. Seed 0.25-0.5 ml of the clarified transfection medium (P0 stock) onto a 25%-50% confluent monolayer of Sf9 or Sf21 cells in 75-80 cm2 flasks containing 15 ml of TC-100 medium supplemented with 10% heat inactivated Fetal Bovine Serum and antibiotics (or Serum free media if preferred). Leave one flask as an uninfected control. Incubate at 28oC and check the growth of the monolayer daily. Initially, there is not enough baculovirus to infect all the cells in the monolayer. Therefore, during the first 1-3 days a growth of monolayer is observed in infected flasks similar to the uninfected control. After 2 days of incubation the first virus buds from the infected cells and eventually all the cells become infected. Infected cells do not divide, but increase in size and often appear granular under the microscope. Contingent upon condition of the cells, medium and initial monolayer density, after 2-3 days of incubation monolayes may be overgrown and there is no apparent difference between transfected and control monolayers. In this case gently resuspend cells from transfected and control monolayers, seed cells from each transfection and control into 80 cm2 flasks and continue the incubation. Harvest the virus stock when about 50% of the cells (25%-75% is also acceptable) collapse as a result of virus infection, typically 5-7 days post infection. Poor virus growth results from poor cell growth – always set up the uninfected cells control and investigate the quality of the cells or medium if the uninfected cells do not grow to confluency. To harvest a successful virus amplification, transfer the cell culture medium into 15 ml conical tubes, centrifuge at 1,500 r.p.m. for 15 min in a swing rotor table top centrifuge and collect the supernatant. Typically the virus titer varies 0.7x108 to 1.5x108 p.f.u./ml contingent largely upon the density of the monolayer at the harvest (50% confluent or subconfluent correspondingly).
The P1 virus stock can be stored for about 3 years at 4oC without practically any loss of the titer. Avoid unnecessary prolonged exposure to bright light as baculovirus stocks are light-sensitive. See more for troubleshooting.
High titer recombinant baculovirus stocks can be obtained either on synthetic serum-free media (Insect-xpress) or on serum-supplemented media (TC-100) using this protocol. Cell growth and virus production times may be different on other media, thus cell seeding densities and incubation times may need to be adjusted. Be aware that stocks produced on synthetic media quickly loose their infectious activity during the storage. The stability can be improved by adding heat inactivated serum to 2% concentration.
Generation of large amounts of high titer recombinant baculovirus stock (P2). P1 stock is sufficient for initial protein expression studies. However, you’ll often need larger amounts of virus stocks to produce significant quantities of recombinant proteins.
Grow Sf9 or Sf21 cells in suspension on TC-100 medium supplemented with 10% heat inactivated FBS or on synthetic Insect-xpress medium supplemented with 2% heat inactivated FBS. Other media could be almost certainly used, but they were not tested in our lab. Grow suspension culture to the cell density 3-5x105 cells/ml or dilute a more dense log phase suspension culture to this density by adding fresh medium.
Infect cells by adding 1 volume of P1 virus stock directly to 1000 volumes of the cell culture. Incubate at 28oC and count the cells daily using hemocytometer. Initially, less then 10% of the cells will be infected as there is at least 10 times more cells then the virus particles. Uninfected cells will continue dividing and after about 36 h of incubation the cell density should reach approximately 1x106 cells/ml. After 24 h of infection the first virus particles will be released from the infected cells, and after about 36 h there should not be any further increase in cell density as infection arrests cell division and all the cells in the culture become infected. Though infected cells do not divide, they increase in size. Amount of cells remains constant for about another 2 days, after which collapse of the cells begins due to infection.
Harvest virus when about 50% of the cells collapsed, which typically occurs 5-6 days post infection. Remove the cells and cell debris by centrifugation at 1,500 r.p.m. for 20 min in JA-10 rotor of J-21 series Beckman or similar centrifuge. Alternatively, a table top centrifuge can be used. The clarified cell culture liquid (P2 stock) may appear a little turbid, that’s normal. As a rule, the P2 sock virus titer is 1-1.5x108 p.f.u./ml. Usually we generate about 200-500 ml of the P2 stock. Storage requirements are the same as for P1 stocks.
|Technology - Maintaining cells - Transfection - Baculovirus stocks - Protein expression|