Interesting question, to which I do not know the answer. But here are some considerations.

Packaging efficiency is related to virion transcript length. If you make a construct that is too large it will not package efficiently. This limits the payload size and unless you are specifically looking at the effect of including introns, you would not have room to casually keep them in. If you are constructing a lentivirus with introns (why?) I imagine they will be spliced, and, depending on the size of the construct, the spliced construct would package more efficiently. Then we'll be balancing the efficiency of splicing with the efficiency of packaging, but probably making a little of both.

Sorry there is no clear answer there, just some early morning ruminations on the topic.

Hi Jason. Thanks for your input. Yes, I agree, packaging limits are a major issue for virus production. The introns in questions are small (100-500bp), so not an obstacle in terms of payload size. True that many introns are >1kb, and so could be problematic.

The rationale for using introns is that large intron-less sequences do not express well (even accounting for lower viral titers with larger cassettes). I attribute this to the fact that non-spliced mRNAs are more likely to get degraded than spliced ones.

I have seen introns used in lentiviruses, for example in the EF1alpha promoter, and synthetic intron in IRES-puro cassettes. However, it isn't clear to me whether these survive in the final lentiviral transcript, or whether they are spliced prior to packaging, and exert their benefits solely through enhanced production of viral RNA in the packaging cells.

Yes, introns are spliced out (the % of spliced transcripts depends on the strenght of your splicing signals, for example beta globin intron 2 is 100% spliced out) , unless you insert the transgene cassette in opposite direction to the viral transcript. In this case the viral transcripts won' harbor the splicing signals and u will keep ur intron in target cells.

I guess you could attempt to isolate the viral RNA from mature virions, RT, and qPCR with a primer starting in the intron and another pair outside of it completely for comparison? Seems like a small hassle to answer an interesting question.

Hello Daniel,

Yes, the introns will be spliced during the packaging. I did this error with a reporter that I want to stably expressed using retro-virus. I got finally a population of virion with a mixture of cDNA coming from all spliced products and pre-mRNA. The potential alternative is to subclone the construct and specific promoter in the oposite sens.

Hope it will help,

Eric

Thanks, Eric. Quite informative. I've seen some folks also try in vitro transcription of lentiviral constructs followed by RNA transfection into packaging cell lines, albeit for other purposes. Did you ever consider this approach? I don't have any experience with IVT and have been leery of low yields with this methodology.

I agree with Annarita Miccio. See, for example, "β-Globin LCR and Intron Elements Cooperate and Direct Spatial Reorganization for Gene Therapy", Buzina et al., PLoS Genetics, 2008. They inserted an intron-containing gene in the antisense orientation in order to improve expression in the target cells, in part because the intron contained signals that enhanced cell type-specific expression in their target cell.

Thanks for the reference, Max!