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[MDavies]

Hello all,

We have a problem which is peculiarly confusing because it affects one of our constructs, but does not affect other constructs which might be expected to be more difficult to deal with.

We have pLVX-puro vectors containing genes for [mCherry], [mCherry + a 3kb protein], and [mCherry + a 2kb protein], expressed from the vector's CMV promoter.  For virus production, these were transfected into 293FT cells along with the rest of the Lenti-X HT packaging mix.  About 80% of the packaging cells were red during the packaging process.

Then when the virus supernatant is titered in 293T cells by puromycin resistance, it's >10^6 per ml, for all three constructs.

However, when the pLVX-puro-mCherry virus supernatant is titered in 293T cells by fluorescence, it doesn't show any fluorescence!  Meanwhile, under the same conditions, the virus supernatants for the fusion constructs are both at >10^5 per ml, measured by fluorescence of 293T cells.

So one of our three constructs seems to express its fluorescent protein from the plasmids transfected into the packaging cells, but not from the DNA copies that result from transducing cells (293T, lymphocytes) with lentivirus containing the same construct.  We've observed this multiple times.  Why would this happen?  

One idea could be: in the packaging cells the plasmid promoters have not had time to be silenced epigenetically, while in the cells used for titering of lentivirus the plasmids are dividing along with the cells' chromosomes and may be more tightly packaged into chromatin.  But why would this only happen with one construct, not the other two?  mCherry alone is certainly not going to be more toxic to cells than an mCherry fusion protein, is it?  So there's no reason why the cells would strive to not produce this protein.

All three look similar in terms of how brightly red the packaging cells are.  And they generate similar titers as measured by puromycin resistance, and as measured by p24 also.  But I am confused that a vector can express its gene in packaging cells, and be successfully turned into infectious lentivirus (conferring puromycin resistance), and yet not express its gene of interest.  I'm especially confused that it is happening with a gene that should not have any concerns about toxicity.

Thanks,

Michael Davies and Shushen Xu
(University of Pittsburgh)