Dear Melanie,

Cells expressing GFP testify that the GFP containing plasmid has gone in, but this doesn't say anything about the other two plasmids, so yes, you can have fluorescence without cells containing all neccesary viral sequences for expression and proper packaging.

There are published data showing that you mostly get co-transfection of two plasmids (provided you pre-mix the plasmids before adding the transfection reagent or nucleofecting), but the co-transfection efficiency goes down the more plasmids you add.

You may find these papers informative:

http://www.ncbi.nlm.nih.gov/pubmed/21081918

http://www.ncbi.nlm.nih.gov/pubmed/19953668

To adress your implied problem of not getting any AAV out of your transfection:

I have not worked with AAV myself before. However, I remember a colleague of mine having difficulties with AAV production and it turned out that the sequence of the ITRs were mutated when she made the plasmid Giga preps (I think they are prone to this as the commonly used E.coli strains do not like the sequence. I have also seen particular bacteria strains advertised that promise to avoid hypermutability in virus preps).

It is difficult to sequence the ITRs due to high GC content, but not impossible.

Hello Mel,

The best example to answer your question that I can think of is: transfect only with the GFP vector, without the Helper and the Rep/Cap, and your packing cells will become green, without the capsid being formed.

I have had GFP-positive 293T cells which never gave a good infection, so I asume that the GFP is not final. However, I must say this was an isolated case, and most often a good GFP signal means that the infection will go right.

Hope this helps

Hi Melanie, 

I agree with Pablo, taking controls along side would be very helpful to address the question. On the other hand correct me if I am wrong but, normally if the GFP is fused with the gene of interest then I believe GFP expression does reflect total expression of the gene. 

Good luck, 

Dear Melanie,

Cells expressing GFP testify that the GFP containing plasmid has gone in, but this doesn't say anything about the other two plasmids, so yes, you can have fluorescence without cells containing all neccesary viral sequences for expression and proper packaging.

There are published data showing that you mostly get co-transfection of two plasmids (provided you pre-mix the plasmids before adding the transfection reagent or nucleofecting), but the co-transfection efficiency goes down the more plasmids you add.

You may find these papers informative:

http://www.ncbi.nlm.nih.gov/pubmed/21081918

http://www.ncbi.nlm.nih.gov/pubmed/19953668

To adress your implied problem of not getting any AAV out of your transfection:

I have not worked with AAV myself before. However, I remember a colleague of mine having difficulties with AAV production and it turned out that the sequence of the ITRs were mutated when she made the plasmid Giga preps (I think they are prone to this as the commonly used E.coli strains do not like the sequence. I have also seen particular bacteria strains advertised that promise to avoid hypermutability in virus preps).

It is difficult to sequence the ITRs due to high GC content, but not impossible.

GFP is in the viral genome, but it will depend upon whether it is inserted before/ after the sequences needed for encapsulation. Anyway transfection of more plasmids may have different optimisation rates for transfection and expression capacity may be different, so the overall result which you are looking for may vary.

As it is mentionned above it does not mean that your virus is well packaged if you see GFP expression, it just mean that your cells express the plasmid. I use the  two plasmids transfection method and my fluo plasmid is the one encoding for rep/cap. To control transfection efficiency (meaning to control the amount of cells that have been transfected) i use flow cytometry, if you get less than 80% of viable cells expressing the plasmid you might get poor titer.

It is in fact really important to digest the ITR regions of your plasmids, use SMA1 for exemple. It highly impact on your viral prod efficiency.

Hope it was helpful

Thank you all for your replies !

One of my colleague found out that this leaky expression of GFP might actually testify of a poor transfection and that the more GFP is expressed the less packaged virus we will have. 

Yes Melanie! This might be true if and only if your GFP plasmid possess the ITR region enabling viral packaging.

In that case do you think that digest the ITR region would improve transfection efficiency ?

Melanie,

Test digests on your ITR regions in the plasmid are to show if these are mutated. SmaI is a restriction endonuclease. So if you loose the particular restriction sites, your ITRs are not the right sequence = bad virus production.

The digestion process is only there to make sure that you have no mutation these sequences as they are so important for packaging. If your ITR are mutated you will make empty capsids only, sad story...

Once you will know they are correct, which in my opinion is extremely important, you can try to improve your transfection protocol.

I have attached a file of some digestion profil that I did in the past, you can observe that on the first lane the profile is not correct compare to the prediction profile (given by my software). Therefore, I reamplified another bacterial clone for this construct.

Personnaly I use Serial Cloner to predict my digestion profile, but I guess many other software exist also.

The prediction profil was done by selecting what enzyme I will use  on my  plasmid nucleotid sequence.

Beside this, If the GFP cassette is "inside" the ITR regions of your plasmid, you might not (or few) observe some GFP signal by flow cytometry, meaning that your virus is well packaged. But as your colleague said, some residual plasmid (not packaged) may however be express by the cells, giving some gfp signal.

I

Thanks a lot this is very helpful !

I think I'm gonna consider indeed to check my ITRs on the GFP plasmid.

Thanks again !

Atb

Mel