During lentivirus packaging, how much of a fluorescent insert is expressed from the viral LTR vs. insert promoter (e.g. CMV)?

02 February 2016 2 5K Report

Hi all,

I'm currently packaging a lentivirus with a large, fluorescently-tagged insert. My gene of interest (GOI) + fluorescent tag is downstream of a tight tetracycline-regulated CMV promoter.

When packaging a lentivirus in HEK293T cells, fluorescent inserts can usually be observed 12-16 hours after transfection of the expression/packaging/envelope proteins in my hands. These are usually downstream of "normal" CMV promoters, and not variants that require transactivation.

Viral RNA is also being transcribed from the LTR on the expression plasmid, and the viral genome will contain the same ORF encoding my GOI + fluorescent protein.

In my current packaging cells, I'm observing no/low fluorescence.

So how much of the fluorescence observed in my cells post-transfection is expected from the transfected plasmid (driven by CMV) vs. expression from the transcribed viral RNA (driven by the LTR)?

I am curious because the tet-regulated CMV should not express much in the packaging cells, but translation from LTR-driven viral RNA would be unaffected. Hence, any fluorescence would be due to the viral RNA.

Thanks!

Abdellatif Benraiss

Hi,

This is all normal. If your expression cassette involves any florescent protein under any promoter (TET inducible or tissue specific promoter) you should see some fluorescence. The reason is that lentivirus is it self driven by strong promoter to express the RNA. That is, the packaged viral RNA span from 5'LTR to 3'LTR and since there is no promoter in LTR (BTW, the promoter which in the 3'LTR has been removed for safety reason since the first generation of lentivirus) a secondary promoter upstream 5'LTR is required for the expression of the viral RNA. Some RNA do not make it to the cell membrane to be packaged, it's translated to protein like any other RNA. Thus the fluorescence you're seeing! I hope this help.

Abdel

Brian Stoveken

Thanks, Abdel. Indeed, I did see strong fluorescence after about 36 hours. This is just much slower than I'd expect to see after transfecting the packaging cells, so I suspect that most of my fluorescence is coming from the LTR driven RNA, without much contribution from the internal tet-regulated promoter. Interesting observation, if nothing else. Im glad the viral RNA is being epxressed appropriately.

Cheers

Gel electrophoresis result for total RNA samples, 1.5 Agarose gel was used, How to improve the integrity of RNA?

Does anyone have a recommendation for yeast reporter gene plasmid/protocol?

Why might my GFP tag be dissociating from my plasmid after transfection?

Expression cloning by using cDNA,do I need to modify the cDNA first?

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

PUC19 as a transfer plasmid for lentiviral delivery?

Any specific pipeline or parameter to analyse total transcriptomics study of brain tissue samples?

Why is the efficiency of viral mRNA electroporation in PBMC low?

Did anyone ever try using pLVX-TetOne Plasmid with a copGFP as a selection marker instead of Puromycin?

Has anyone had luck using cell lines other than HEK 293 (such as COS1) to package lentivirus?