Can you describe the two lentiviral constructs in regards to the position of each of the elements in them (i.e. promoters, GFP, IRES, antibiotic resistance genes, etc)?

Thanks for the reply Tom Masi

The first construct delivers the PylRS tRNA(CUA) expression cassette. In this construct, 4x tandem tRNA(CUA) ORFs are expressed from 4x h7SK promoters. In the opposite direction, the PylRS ORF is expressed via an EF1 promoter followed by an IRES and puromycin resistance marker.

The second construct delivers a reporter, namely sfGFP containing an in-frame TAG mutation. In this construct, sfGFP-TAG is expressed via a CMV promoter; an IRES and blasticidin resistance marker follow the sfGFP-TAG ORF.

I've attached graphical representations of both constructs as well.

Thanks Ryan. Perhaps you can explain how GFP would be responsive to the addition of UAAs when the CMV promoter should be driving expression of GFP based on the construct maps?

Tom Masi

Absolutely. The GFP ORF contains an in-frame Amber stop codon (TAG). So under normal conditions, the translation of GFP is aborted prematurely. However, in the presence of UAAs compatible with PylRS and tRNA(CUA), the Amber stop codon recruits the tRNA(CUA) and the protein is made. Let me know if this is unclear!

Thanks Ryan. Very clever reporter I must say! I agree with you that your problem is transduction related or more specifically, an issue with integration into the genome. Either your PyIRS or GFP has been partially deleted/deleted/mutated such that it is not functional after translation. Obviously you are getting expression of the antibiotic resistance genes but those are translated independently of the other ORFs. You may want to PCR amplify PyIRS and GFP from the genome and sequence them which might tell you what the problem is.

In regards to the 1% GFP expression you observe, this is probably read-through of your TAG stop codon. Not at all unheard of. Whenever I make a construct where I express/purify proteins, I always put 2-3 TAGs at the end such that read-through does not occur.

When you transfected the GFP/UAA utilization plasmids into HEK293T cells to confirm the system works, did you use the lentiviral transfer plasmids?

Tom Masi

Thank you for your insights, they are very helpful! I can't take credit for the reporter, I'm following what a previous group has already done (Article Generation of influenza A viruses as live but replication-in...

Perhaps the most perplexing observation so far is the fact that the plasmids work as expected when transfected into HEK293T cells (see attached image).

With all of this in mind, I think we've narrowed down the source of the issue to transduction/genome integration. My current plan to resolve this issue is to package lentiviruses again, concentrate them, and transduce cells at a range of high MOIs. If the issue is with low PylRS expression, this could serve as a solution. I figure the transfection results strongly support this hypothesis - there are likely many copies of the PylRS gene being expressed after transfection but maybe only one or a few copies being expressed after transduction. What are your thoughts?

I don't find it perplexing when the plasmids are just transfected into HEKs that the system works. When this is done, the system is being run off the plasmids themselves while when you use the lentivirus the system runs off the integrated copies in the genome which can have issues during integration. The transfection is a good positive control. The other thing is with the lentivirus you are selecting for stable integrants while I am assuming when you just transfect the plasmids you are not doing antibiotic selection?

Is there a particular reason you are using lentivirus to get your system into the HEKs and not just transfect the plasmids and select from there? I ask this because if you are getting good transfection of the plasmids needed to make lentivirus, you should get good transfection of just the plasmids containing the constructs and then select for double stables from there?

I wouldn't expect that you would have to use an MOI greater than 5 to get close to 100% transduction efficiency of HEKs with lentivirus. They are highly transducible. Plus too high of an MOI leads to cell death and other problems for the cells. I work with human stem cells and an MOI of 5 gets me >90% transduction efficiency which is good enough because you will never reach 100% by increasing the MOI. Trust me, I've tried it.

Tom Masi

Your assumption is correct - when I transfect, I don't select with antibiotics.

The reason for using lentivirus is two-fold: 1) we want to have stable lines for rescuing influenza virus from DNA (8 plasmid system), so not having to transfect an additional 2 plasmids would be optimal (but not necessary I suppose) and 2) our rescue system requires MDCK cells to amplify the virus produced on 293T cells - to my knowledge MDCKs are not very transfectable, which is why we opt for lentiviral transduction.

I could try generating a stable 293T line via transfection; if that works, then I have at least 1/2 of the puzzle solved. Might be worth trying with MDCKs too. Will probably have to troubleshoot the transfection a bit to maximize integration events. What do you think?

Thanks again for the discussion, this is super helpful!

Thanks for the information. Yes, don't mess around trying to transfect cells that are not highly transfectable. If you don't already do this, I would suggest making a GFP lentivirus as a positive control along with your other lentiviruses. That way you will be able to monitor all aspects of the lentivirus production (cells should glow green after transfection). Plus, after you concentrate the virus you can titer using the GFP lenti and your other lentis should be of similar titer. Then you can try different MOIs using the GFP lenti and see what gives you >90% efficiency.

What sort of scale to you make your lentivirus (100mm dish, etc.)? How do you concentrate the virus?

Ah, that's a very clever and easy way to gauge titer. I will do that.

Normally, we package lentivirus in a 6-well plate and use the entire ~1.5 mL to transduce a 6-well of our target cell line. However, when I do large scale productions, I use 1-3x 15-cm dishes.

In the past, I have concentrated virus using Millipore Amicon centrifugal filter units but going forward I plan to use a chemical-based approach (Takara Bio's Lenti-X concentrator; I think it's PEG or something similar).

So you think if I can achieve ~90% transduction efficiency, my reporter system should start functioning appropriately?

I think I have discovered the issue. I performed parallel transductions using lentiviruses packaged with mCherry to gauge transduction efficiency. With 293Ts, I identified a dose of lentivirus that yields >90% transduction. However, after selection, these cells remain unresponsive to UAAs and do not produce GFP. Thus, expression of the PylRS genes is likely not the issue here.

With these results in mind, I began to wonder if the 4 tandem repeats of tRNA cassettes would present issues during packaging or transduction. I hypothesized that, given the repeating sequences, this region of DNA may be subjected to excision via homologous recombination or some other process. To test this idea, I excised this region from my Pyl-tRNA-PylRS expression plasmid using restriction enzymes. Then, I transfected this DNA into the transduced 293T cell line that contains the PylRS genes but is unresponsive to UAAs. To my surprise, in the presence of UAAs and linear tRNA cassettes, these cells are able to produce GFP.

These results suggest that the repetitive tRNA expression module is somehow compromised during lentiviral packaging or transduction steps.

Thank you for your help Tom Masi, your feedback helped me arrive at this conclusion! I hope this is useful for anyone else experiencing this issue.