Dear colleagues,
We need some suggestions about baculovirus system. We utilize Bac-toBac HBM secreted system (Invitrogen to express our protein). For transfection Cellfectin 2 is used.
The problem is the following: after transfection of Sf9 cells with the bacmid cytopathic effect (CPE) becomes evident only 2 weeks post transfection, not within 5 days is we expected.
Cells transfected with the control bacmid (derived from pFastBac™ Gus plasmid, carries beta-glucuronidase gene, supplied with the kit as a control) develop pronounced CPE in 5 days post transfection.
Our product appears in medium only at day 9 post-transfection, while Gus becomes detectable in supernatants at day 5.
GUS gene is 1.8 kb, our gene is 2.5 kb.
Both bacmides were purified simultaneously with phenol-chloroform extraction, concentrations were measured with QBit. We have tested various Cellfectin:DNA ratios – generally nothing changes.
Thus, obviously transfection efficiency with our gene is much lower.
What is the possible trouble? Something is wrong with bacmid? Gene is non-toxic for mammalian cells (in our hands) and for insects too (according to literature).
Any suggestions will be really valuable,
Thank you in advance.
As your product did appear in the medium(double check it), although a little late, it means your protein was expressed well but expression level is low.
But you know, the P1 viral stock is a low-titer stock. I would suggest you amplifying your stock first, and then do the expression with with a suitable high titer(e.g., 10e8pfu/mL).
If it still doesn't work, you can also try to express your protein using non-secreted system, in which you may have a better expression level sometimes.
Good luck!
Try lipfectamin or Roche x treme reagent , might give you better results with less amount of transfection reagent
Each protein expression has different pattern so harvest the cells when your protein is better expressed. ( when using MultiBac the YFP gene tells you when the virus is expressing the gene better but still we have to check via SDS PAGE at different DPAs)
Are both the promoters in the bacmid under which the genes ( GUS and your GOI) are present the same?
some other factors to look out for are:
The health of the cells at the time of infection ( how many times subcultured)
The amount of cells used ( usually 0.5 million cells per one well of a 6 well plate ) is a good amount for infecting using upto 10ug of Bacmid.
The incubation time of bacmid with the transfection reagent - 15 or 20 min
The media temperature and no FBS helps better transfection
and also as suggested by Lin Bai, The V1 stock is better for large scale expressions
hope this helps
best
Dear colleagues, thank you for your suggestions. Lakshmi, answering to your question: yes, promors are the same for GOI and GUS (control gene). I understand that expression of GOI may be much lower than of control gene, especially in a P1, that's no suprise. We are not trying to purify protein from P1 supernatant, for sure virus will be amplifird. What is not clear to me: I have two bacmids, at the same concentration. When I transfect cells with control bacmid CPE is seen quite early, as it is described in manual. But during transfection with bacmid with GOI in the same conditions CPE is delayed. Is it possible that insert influence somehow on virus amplification/infection rate? Does it make sense to try bacmids isolated from several colonies?
Sorry for the delay in reply. The protein expression can vary from clone to clone or virus to virus .Usually ( I used MultiBac system) I transfected 2 or more bacmids isolated from different white colonies in 6 well plates (Sf21 cells, around 0.5-1 mill/well) checked the virulence( when infecting V1 to 50ml cultures, their infection efficiency ) and protein expression( from 6 well plate, western blot ).This kind of helps early screening of defective virus and protein expression.
Hope this helps
I can not see the bacmid cytopathic effect (CPE) even 7 days post-transfection with Lipofectimine 2000.The bacmid is 300ng/ul,can anyone give some suggestions?Besides,what is the minimal concentration of the bacmid for transfection and how to achieve the high concentration of the bacmid?Thanks.
Hi Xiaoqiang
Sometimes the bacmid transfection was low efficiency hence there was no CPE even after 7 days. If you do not see the effect within 48-72 hours it is always better to restart the infection with fresh batch. Please check the lipofectimine protocols for optimal concentration, but I recommend is to make a overnight culture of the bacmid containing bacteria( hope you are doing the recombination in Bacteria), 2-5ml culture use it for bacmid prep and use it all for transfection into two wells ( each clone) by mixing with transfection reagent and media ( Serum Free). The incubation time and mixing matters here. Also treat the bacmid gently since it is a big DNA molecule if you pipette it a lot it will be sheared. Hope this helps
Best
Dear Vijayachandran,
Thanks very much for your professional advice.I did the the recombination in bacteria,as suggested by the Bac-to-Bac manual.Besides Cellfectin,have you succeed with other transfection reagents?And is the Bacmid high-copy or low-copy?Thanks.
Hi Xiaoqiang,
I have used Roch Transfection reagent X treme and also lipofectimin. They both work well .Usually Bacmid is only single copy per bacteria ( I know about MultiBac better)
Some points to keep in mind are
The DNA: Reagent ratio
The time of incubation for the reagent mixture
The cell density in the well
Healthy cells
Fresh DNA preparation
Best
Lakshmi
Dear Vijayachandran,
Again,thanks very much for your kind explainations.Bacmid used for transfections is freshly prepared and the sf9 cells are low-passage healthy(>95% viability)ones, with 1 × 106 cells/well in a 6-well plate.Could you please send me your detailed protocol(expecially the DNA:reagent ratio and incubation time) to me at [email protected]?
Thanks.
Best
Xiaoqiang
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