For sf9 vector transformation, almost all literature suggests to transform it when the cells is in adherent form. my cells are suspension in grace insect medium supplemented.
Would you please inform me if it affects the efficiency of baculovirus vector transformation? Specially my gene is large (3000 bp)
Thanks
Hi Shiva,
Sf9 (along with Sf21 and High5s) are happy switching between suspension and 'adherent' culture. You can transfect them in either state without any problem. The main reasons for using 'adherent' cells is the small volumes of cells and DNA is easier to be used. It is also a bit easier to monitor the infection process once recombination has occurred. There is also a certain amount of not changing what's working as this was the original methodology.
If you are using the recombination in insect cell methods I would recommend using the cells in 'adherent' form as the efficiency of recombination is low and it is easier to follow that the recombinant virus has been generated.
If however you are using the bac-to-bac system where it is much easier to generate more DNA transfecting the DNA into 5-10mls of cells in Tubespin tubes works well and leads to a larger p1 stocks. see method:
1. Count mid-log Sf21 cells with the CEDEX. 2. Place 300µl of Media in a Tubespin tube. 3. Add 40µl of Superfect reagent to the media. 4. Add 10µl of the bacmid to this mix and incubate for 30 minutes at room temperature. 5. Transfer 5*10E6 cells (per transfection) to a centrifuge tube and centrifuge at 300*g for 15 minutes. 6. Remove the supernatant and resuspend the cell pellet in Media in 1,7ml times the number of transfections (10 transfections = 17ml). 7. Transfer 1,7ml of this cell suspension to each Tubespin tube, which now contains around 2ml of cell suspension with 5*10E6 cells in total. 8. Transfer Tubespin tubes to the 27°C incubator and incubate for 4 hours. 9. Add 8ml of Media and incubate for up to 7 days (or until viability of cells has dropped below 75%). 10. Monitor cells for signs of infection using cell number, viability and morphology through a daily cell count. (Note: if using media with FBS to culture the cells only used FBS in step 9)
-Richard
Note: The cells are not really adherent, they are just resting on the plate bottom and are slightly sticky and easily dislodged.
I agree with everything Richard says. I have tried a couple of protocols for transfecting in suspension and it seems to work equally well as in adherent.
If you have access to a cell counter that also measures cell diameter (I use a Countess Cell Counter, which works well as long as cells are not too clumpy) it is much easier to follow the transfection. As well as seeing when growth stops, you can chose to harvest V0 (or Baculovirus Infected Insect Cells (BIICS)) when your cells have reached a certain size (e.g. a 3µm increase, but very dependent on cells and growth conditions) whilst keeping an eye on viability.
Thanks for your answers. Also would you please mention the concentration of bacmid.
Thanks
Hi Shiva,
the concentration of the bacmid is very difficult to obtain (and not really needed) due to contamination from other plasmids.
The above method uses bacmid preped from a 2ml overnight culture by standard alkali lysis followed by Ethanol precipitation resuspended in 40ul H2O or TE.
-Richard
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