No, introns are not retained with measurable frequency in lentivirus. The only way to achieve this would be to perform in vitro transcription of your intron-containing lentiviral RNA genome, and then transfect this RNA into a 293T packaging cell line.

Nonetheless, viral-mediated expression is typically quite strong, and the WPRE element allows nuclear export of unspliced viral-sourced mRNAs. Unless you are trying to express a large cDNA (>= 2kb), or look at rare cases of intron-dependent gene expression (e.g. XBP1),  I'm not sure there would be a good reason to try and keep an intron for typical transgene delivery. Are you sure it is necessary for your experiments?

An intron can be retained in a lentiviral vector. The trick is to provide the sequence in the other strand as the one that is used for viral packaging. Most lentiviral vectors rely on a promoter (often the CMV promoter) upstream of the 5' LTR to drive expression of the viral RNA genome; I will call this strand the positive strand now). If you put an intron in this sequence (as in: the positive strand in this case), it will be spliced out during packaging and you'll end up with a shorter viral genome and hence shorter integrated product. If you clone the intron in the negative strand (that is, the splice donor and acceptor sites are in the negative strand), this intron is retained in the virus and also incorporated in the genome. Hence, the pol-II promoter to drive expression of your gene of interest (with the intron) should be in the opposite direction as the pol-II promoter which is used for the packaging... 

I've tried this, and it works. However, you often get lower virus-titers since you get issues with transcriptional interference due to the opposing promoters... The drop is ±5 fold in our hands.

Thank  you, actually an intron is not strctly necessary but I had reasons to retain it. I'll consider if antisense insertion may be feasible.

True, an antisense cassette can in principle retain introns in lentivirus, but the drawbacks of low titers, often outweigh the benefits. Low titers caused by an antisense promoter will be further compounded by increasing the payload size of your transgene. Total insert size has a large effect on lentiviral titers.

That is true indeed. But for some cases it can be beneficial to include introns in a lenti. For instance, we use introns to express clusters of microRNAs in cells. Normally, when expressing microRNAs from a 3'UTR, the upstream gene is expressed at low/undetectable levels since the mRNA is destabilised by processing out of the miRNA from the 3'UTR. When you place the miRNAs in the intron, the reporter gene is highly expressed and you have high expression of the miRNAs in question. It all kinda depends on the question the researches has. In most cases, I would also not use an intron in a lenti.

Excellent point, Robert. I hadn't considered the miRNA application in my responses.

Indeed, in some cases canonical nuclear processing can be important for proper mRNA metabolism. By the way, I am working on mRNA targeting..

In that case, I think all bets are off. No way to know if mRNAs that normally have a specific form of subcellular targeting will still exhibit this behavior when expressed off a viral cassette. You'll have to test it empirically.

We have found that introns can be maintained and packaged although at varying efficiencies. To be on the safe-side we tend to put the cassette in the reverse orientation as suggested above to ensure it is maintained.  If doing this, don't forget you'll need a complete cassette - a promoter and of course a polyA signal at the end of your reverse orientation gene.  Also, just be aware that anything you do insert in a reverse orientation may cause problems if read in the sense orientation to produce the viral genome (such as a polyA site which would terminate your vector genome and prevent packaging).  It would be unlucky, but if you get zero titre, that could be a reason why.

To help negate some of the problems of a reduced titre, try co-expressing NovB2 (see papers by Tobias Maetzig et al when working in Axel Schambach's group) as this can reduce the effects of what is essentially RNAi caused by the reverse orientation cassette.

Hope that makes 'sense'.  

Thank you all for your help. Steven, why do you think  a polyA in the reverse orientation would affect packaging (apart from dsRNA degradation of course)? It would be more of a trouble in the sense one. Anyway I am quantifying titer as effective infecting units, and via RT PCR on a sequence at the very 3' end of my viral genome, so I'll keep you updated of my results. 

Sorry Francesco, I didn't make myself clear.  The polyA signal in the reverse orientation should not affect packaging (unless it is palindromic and can read in both directions).  What I was trying to say is if you are unlucky, when you insert a cassette in backwards, there may be sequences in the cassette's antisense strand (i.e. the 'sense' strand for the viral genome) that by chance act as PolyA signals (or splice sites etc) and interfere with the production of the virus.  I think this is a rare event, but I was just suggesting that if you do try the reverse orientation trick and get no virus, check there is nothing on the antisense strand of that reversed cassette that would interfere with the production of viral genome.  This is much easier to explain with a pen and paper!  If I'm still not making sense, please let me know and I'll try to upload a picture.

This is an excellent thread. Robert, I found this paper http://advances.sciencemag.org/content/2/10/e1601737.full  that has a similar design to what you indicated. What do you think would happen if the Intron is put in a "sense" orientation instead of antisense?

Dear Colleague,

I hope this message finds you well. Your query regarding the retention of introns in lentiviral vectors is indeed an insightful one, touching upon the nuances of molecular cloning and viral vector design. Here, I aim to provide a comprehensive overview that addresses your question with the clarity and depth it merits.

Lentiviral vectors, derived from the Lentivirus genus, have become a cornerstone in gene therapy, transgenic models, and functional genomics due to their ability to efficiently deliver genetic material into the genome of dividing and non-dividing cells. One of the key features of these vectors is their ability to accommodate relatively large genetic payloads, including complex regulatory elements, which makes them highly versatile for various applications.

Regarding the specific question of intron retention, lentiviral vectors are typically engineered to carry a transgene expression cassette that lacks introns. This design choice is grounded in several considerations:

However, it's worth noting that there are specific scenarios where incorporating introns into lentiviral vectors might be deliberately chosen, such as when aiming to enhance the expression of certain transgenes. Some studies have shown that the inclusion of a strategically placed intron can increase mRNA export from the nucleus, improve mRNA stability, and enhance translation efficiency. Such strategies are usually employed with careful consideration of the trade-offs mentioned above.

In summary, while the default engineering strategy for lentiviral vectors is to exclude introns from the expression cassette to maximize efficiency and stability, there are nuanced exceptions where introns may be included to achieve specific scientific objectives. The decision to retain or omit introns should thus be informed by the specific requirements of your experimental design and the characteristics of the gene of interest.

I trust this explanation provides a clear and thorough response to your inquiry. Should you have any further questions or need additional clarification, please feel free to reach out.

Warm regards,

Reviewing the protocols listed here may offer further guidance in addressing this issue