After transducing cells with a lentivirus and passaging them with the proper selection drug, can they stop producing a transduced gene of interest? I have transduced cells to express a protein and then selected them in puromycin. It seems that they express the protein less after multiple passages.
Is there a general number of time mammalian cells can be passaged after transduction with a lentivirus?
Is it possible that they lose the gene of interest or that it gets down regulated over time?
If so, how do the cells remain puromycin resistant if the gene of interest and puromycin resistance gene are under the same promoter (CMV), separated by an IRES?
Hi,
perhaps you have a silencing of CMV promoter during the time. Have you keep cells under selection ? What is the position of your gene of interest ? before or after IRES ?
The cells have been kept under puromycin and have remained resistant.
The gene of interest is directly after the CMV promoter and before the IRES. The puromycin resistance gene is after the IRES.
Hi David
First about the protocol: when you do a selection with antibiotic after transduction, you have to absolutely continue to cultivate them under selection otherwise you promote events of silencing.
Second about the design: IRES can induce a differential of expression about the two gene. So you can have your gene of selection which is well expressed while your gene of interest is not expressed at all.
To solve this problem, you can use 2A fusion. That's works very well and you have not this type of problem, we currently use it in my lab and we are very satified (many more than when we used IRES)
You can find more information about this type of design hereafter
Article High Cleavage Efficiency of a 2A Peptide Derived from Porcin...
And if you want information about transduction protocols:
https://www.geg-tech.com/knowledge/lentiviral-vectors-multifaceted-tools
Hope this will help you,
Regards
Nicolas
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