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.
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?
Edited by miST32, 11 February 2016 - 09:29 AM.