7.1. Growth conditions for SF9 cells
Insect cells were grown in SF900 II medium without antibiotics at 27°C when not infected and at 28°C when infected with virus. Doubling time of healthy cultures was usually approx. 24h and was determined with a Neubauer improved counting chamber (Peske). Unless for subsequent freezing or in case of slower doubling times or apparent system malfunctions cells were grown without FCS, for bigger expression penicillin/streptomycin was added. Cells were split 1:3 into fresh flasks approx every 2-3 days when culture plate or flask became 100% confluent.
7.2. Freezing and thawing of insect cells
For long term storage and strain establishment, insect cells were harvested at 80% confluence in mid-log phase, centrifuged at 300 g for 10min and resuspended in medium containing 10% DMSO and aliquoted. Subsequently cells were slowly cooled down for 1 h at –20°C before transferring them to –80°C and finally into liquid nitrogen. For thawing, a 1 ml liquid nitrogen stock was quickly submerged in a 37°C water bath until it was completely thawed (1-2 min) avoiding shaking. The culture was quickly pipetted into tissue culture 25 ml flask with 5 ml preheated medium using a sterile plastic Pasteur pipette to avoid shearing forces that would occur with pipette tips with smaller outlets. Cells were allowed to settle during 1 h at room temperature with the colonizable surface of the flask being reduced by creating an inclined plane.
several times on the same day and then once a day until the plate was confluent again and split with normal rates.
7.3. Transposon mutagenesis and blue/white selection
The pFastBac1-CDK8 plasmid was transformed into DH10-BacTM cells. Instead of directly plating the cells after heat shock recovery (in 1 ml SOC medium), 10 ml of SOC medium supplemented with kan, tet and gen were added and cells were grown for 96 h under vigorous shaking (230 rpm) at 37°C before plating them at three different concentrations (1:1, 1:10 and 1:100) onto fresh Blue/White selection plates containing the antibiotics plus IPTG and X-Gal. Plates where incubated over night at 37 °C. Suitable white colonies were picked and replated on selection plates for a color control. A glycerol stock was prepared (addition of 20% sterile glycerol in 1 ml total volume, storage at -80°C) and the DNA was isolated as described above with the user-adapted protocol for MidiPrep with the Qiagen Kit. Success of mutagenesis was controlled by PCR using combinations of M13 primers, CDK8 specific primers, and combinations of both.
7.4. Isolation of Bacmid DNA
For the isolation of Bacmid DNA, a 100 ml culture with selective LB medium was inoculated with 0.5 ml from a 5 ml overnight starter culture of recombinant DH10 Bac cells and grown for 14 h under vigorous shaking (250 rpm). DNA was isolated with the DNA MidiPrep Kit according to a user adapted protocol where the QF elution buffer was preheated to 65°C and elution was performed in five 1ml steps instead of one 5ml step. This leads to a higher yields of high molecular weight DNA, the division in 5 steps prevents cooling of the buffer. Bacmid DNA was resuspended in 100 "l TE buffer and had a concentration of 580 "g/ml.
7.5. Transfection of SF9 cells with Bacmid DNA
15 "l of Bacmid DNA at 581 ng/"l and 18 "l of Lipofectin (Invitrogen) were each mixed thoroughly with 250 "l of serum free medium without antibiotics in sterile tubes made of polystyrene (Falcon). Usage of a different material greatly reduces transfection efficiency as DNA-lipid complexes stick to the reaction tube when this is made for example of polyethylene. The two solutions were combined and incubated
at room temperature for 15-45 min. For transfection 2 ml of medium was added to the transfection mix, an 80% confluent mid-log phase 5 ml insect culture was washed once with medium and overlaid with the mix. Cells were incubated at 28°C for 5 hours before the transfection mix was aspirated off and overlaid with 5 ml medium containing antibiotics. Cells were incubated for 5 days and supernatant was harvested by centrifugation (10 min, 300g, 4°C).
7.6. Harvesting of initial virus stocks and virus reamplification
The 5 ml first generation virus stock was split in two and two 80% confluent mid-log phase 5 ml insect cell cultures were reinfected overlaying the culture with 4 ml fresh medium and adding 2.5 ml virus stock to each flask. Cells were incubated until lysis occurred (5 days) and supernatants were harvested by centrifugation. In a third and fourth step culture sizes were increased by diluting the virus 1:4 and 1:10 respectively and infection were carried out for 48 h. Supernatants from subsequent expression tests (48-72 h) were pooled and used as the working virus stock. An optimal infection ratio of 1:7 where 90% of the cells had detached 24 h postinfection was determined for this stock. Supernatants of subsequent infections were pooled for a new stock. Virus stocks were stored at 4°, where they are stable for a few weeks.
7.7. Expression of CDK8
Expression was carried out applying fresh medium mixed at a 1:7 ratio with infectious supernatant onto an 80% confluent plate that had been split the previous day. The optimal infection time was determined and further expression were carried out for 24 h before cells were harvested by centrifugation at 4°C and resuspended in lysis buffer. Supernatant was removed and reused as described.