Lentivirus packaging (no fluorescence) - (Feb/11/2016 )

Hi all,

Our lab routinely produces lentiviral particles for in vitro and in vivo transductions.  

I'm currently packaging a 2nd gen virus.  The expression plasmid contains a large insert (~4kb) with a fluorescent tag downstream of a tet-responsive promoter, plus a second promoter driving an rtTA cassette.  

Usually I see fluorescence 16 hours after transfection of the HEK293T host cells with a fluorescent viral expression plasmid.  My understanding is that this is due to host cell translation from the LTR-driven viral RNA genome.

Unfortunatley - I don't see fluorescence this time.  

I use 4:3:1 ratio of Expression:Packaging:Envelope plasmid, and 3:1 ratio of lipofectamine:DNA.

My question is if the size of the insert might reduce the amount of fluorescent protein I'd normally see during packaging, or delay it?  I don't need high titer (would be nice), but I'm alarmed to not see the 80+ % fluorescence in my packaging cells that I'm used to.  No fluorescence at all yet, actually.  Very bizarre.

- I'm re-checking the DNA via restriction digest just to make sure I didn't purify useless expression plasmid in the last prep.
- The sequenced plasmid looks good.

Any thoughts?

edit:  One possibility that crossed my mind is the tet-responsive promoter (tight TRE CMV) being poorly active during packaging, and thus the fluorescence would be completely reliant on LTR-driven transcription.  All of my other viruses have had CMV driving the insert RNA as well as LTR-driven viral genome.  Might this reduce the expected level of fluorescence?

Update:

The cells became fluorescent much later in the packaging process than I've seen before.  This seems consistent with either a slower accumulation of fluorescent product (Low activity of the Tet-On promoter, only LTR viral transcripts).  Could also be due to insert size, which is pushing the virus near 9.5kb.  

In any case, the virus seems to work - if others find themselves in this situation there's no need to panic.