Hi Guillaume,

I've some of thoughts.

Generally the bad-to-bac system works well and will generate recombinant virus from the bacmid. So you should be getting virus if you have followed the protocol. 

First infection for virus production p0 to p1 is not optimal for protein expression - although I would expect to be able to see expression. You don't say at what time point you are analysing for expression, is it after several days? You could be seeing degradation of the protein or cleavage of a tag (if that's what you are looking for).

Secondly have you titred your virus as that would help determine if you have a generation problem. Also it's worth checking that the bacmid is not heterogeneous with not recombinant bacmid - there's a PCR method described in the manual.

Thirdly doing a correct expression grow, at MOI=3, POI=3e6cells/ml and time points 48,72 & 96 would be much better way to determine expression than to look in the virus production cells. Personally I never bother checking expression until I have a tired virus that I can use to do a proper experiment - the quality of information it will return is much better for evaluating expression.

-Richard

Hi Richard,

Well I have to say that I'm a novice regarding baculovirus technology. I'm following an empirical protocol established by people like me I'm afraid. There is no titer determination but only a small scale infection with increasing volume of P2 to determine the best quantity of virus to obtain the best protein expression. So it is very basic. After 4 days of transfection in 6 well plates I collect the P0 to infect 5 million cells in T25 flask and leave again for 4 days. At this step I retrieve transfected cells and analyse protein expression which is in my case good. However the cells infected with P0 for 4 days do not expressed any of my proteins. Have, for one of them (GST-tag), what could be degradation. However this degradation is similar whatever the GST-tag protein I'm trying to express.

There might be something wrong with amount of cells and volume of P0 I'm using for P1 generation. I don't know but you may have an idea and solutions.

Guillaume.

Hi Guillaume,

many times baculovirus generation and expression just works, and you don't need to pay too much attention to the details. This however can prove a trap for the inexperienced as when it doesn't work then it can be very hard to work out why. I recommend that you follow the bac-to-bac manual protocols closely, checking at as many points as possible. It also has a good method for plaque assay - this will take some time to get the result but will provide you with good information as to the virus.

Normally it's not practice to titre the p0 from the transfection due to the low volumes, however if you are having problems I'd suggest that you do so. One problem that can be hard to detect without titreing your virus is if you are getting a low titre, the cells will look infected but if you use this virus for p1 generation you will get a low titre again and so on for p2. Expression from this virus will be sub- to very sub-optimal. If you have a low titre, then you can adjust the MOI to generate virus that should be better.

Also spend time getting to know your cells well. How they look & grow when infected. How they look & grow if infected at too low a MOI. 

I'd also suggest you double check that your construct is correct and that there's nothing that's accidentally been introduced into the sequence to prevent the expression.

-Richard

https://tools.thermofisher.com/content/sfs/manuals/bactobac_man.pdf

Hej,

Richard already mentioned some important aspects.

When we started with baculoviruses we faced some comparable difficulties. And I learned that even experienced virologists did not follow án important rule: Always work with plaque purified isolates! Using the bac-to-bac system this is of special importance bcause even if you isolated DNA from a single E. coli colony you can have a mixture of bacmids consisting of molecules with or without insert becaus 4 or even mor bacmids can be present in the same bacterial clon. To make things easier, my lab constructed several bac-to-bac transfer vectors which facilitate the whole procedure ( see: Keil et al,, Journal of Virological Methods 160 (2009) 132–137. If you are interested to obtain these plasmids: just request by e-mail.

Cheers,

Günther

Hi,

Thanks for your advices and sorry for my late answer. I did in the past baculovirus isolation from plaques but this was 20 years ago. I'll have look on your paper Günther. However, what I don't get is why when I transfect 4 different bacmids having the correct insert (PCR checked), all transfected Sf9 cells express the correct protein but nothing in Sf9 infected with P0. What I meant is they express the same nothing so I cannot imagine that the 4 different bacmid prep contain the same bacmid mix and that the empty one is taking the lead over the recombinant...

I repeated transfection and I start a new P1 I'll let you know.

Cheers,

Guillaume.

Hi,

Got he same bad result with my new transfection. Protein is expressed upon bacmid transfection, virus are produced (all signs of infection) but likely not recombinant. Indeed, infected cells with P0 (or P1 according to Invitrogen) are not expressing anything...

By PCR none of them gave me the product for empty bacmid or even bacmids recombined with empty pFastBac so I'm lost.

Günther, I had a quick look on your paper (very cool) but don't understand how your modified pFastBac plasmids are better to generate recombinant bacmids. It's probably because I didn't read it carefully.

Cheers,

Guillaume

Hi Guillaume,

Can you tell us how are you detecting the protein expression?

The only things I can think of right now - and this is pure speculation.

Is that when you transfect with the recombinant bacmid, you are also transfecting with a large quantity of the fastbac vector. Now you won't get any expression from these plasmids until the baculovirus has gone through it's life cycle, but then you would get expression once the virus enters the very late phase. So is this showing you expression as you have a much larger number of copies of your expression cassette? Once you have generated virus subsequent infections will have a much lower copy number of expression cassettes, and your expression level might be below detection level because of this.

The second thing would be to sequence the PCR product from the baculovirus DNA, to check that there's not some rearrangement occurring when you generate the recombinant virus.

-Richard

Hej Guillaume,

the major advantage of the platform I that Generation, growth and titers reached by the recombinants can be monitored 2 to 3 days after transfection/infection without any processing. With regard to your Problem. For me it now appears that the protein you are trying to express is inhibitory  for baculoviruses. Would be helpful to know whether this protein or homologs have been successfully expressed by baculoviruses. We had such Proteins twice and had to shift to vertebrate cells using BacMams.

Best,

Günther

Hi,

Sorry for my late answer, I'll start answering Richard. I'm detecting protein expression from bacmid transfected cells 4 days after transfection when collecting P0 simply by running WCE on SDS/PAGE and Coomassie staining. I confirmed this result by immunoblot using specific antibodies.

I checked all my bacmids by PCR and empty bacmids are not present but regarding pFastBac contamination I don't know. I there a way of checking for its presence? A way of getting rid off it?

Concerning the proteins now, I managed to produce one of them with a different tag (GFP instead of GST) without major issues. Regarding the second one, it allways failed whatever the tag used but this is the first time I'm detecting it in transfected cells. However, it seems mostly insoluble...

Could, Sf9 be a problem? The one I was using were quite old by managed to generate some recombinant baculoviruses for other proteins.

Well hope you'll get some more advices to solve my problem.

Cheers,

Guillaume.

Hi Guillaume,

I'll admit I'm at a bit of a loss as to what to suggest to help you here.

Trying with new Sf9s wouldn't do any harm, and it's good practice to not use cells that have gone through too many passages. However Sf9s are quite robust in working after many passages and I'd be a bit surprised if this is your problem.

-Richard

Hi,

Are your infected SF9s displaying any CPE after infection with P0 stock? You could looking at the cells using IF to see if they are infected? Buy a gp64 mAb and a conjugated secondary antibody and do this. Alternatively you could do a WB with the gp64 mAb?

If you do have baculovirus being made, you can always take some of the growth medium and extract the nucleic acid for PCR and sequencing to check it still has your gene of interest? I've done this before where we were having issues with our expressed protein. I took 200ul of cell culture medium from the infected cells and extracted the nucleic acids using Qiagen's minelute virus spin kit. I can't remember if I used the M13F&R primers normally used to check the bacmid construct or if I used primers specific for my gene of interest. Basically, after amplifying and sending for sequencing we found out there was a mutation in the sequence that caused a premature stop to appear.

John