We have been trying to produce lentivirus with the Tet inducible system for a while, but we are getting no signal in the infected cells after infection. However, after transfection, there is a higher signal in the cells. Has anyone had this problem, or has any explanation for this situation?
Can you clarify what the "signal" is that you are looking for? And is the "signal" under the control of the Tet On promoter (the addition of tetracycline induces expression of the GOI)?
Regarding the transfection, you are transfecting the transfer vector (plasmid) that contains the viral genome/tet inducible system?
Tom Masi The signal is related to the mCherry reporter gene tagged to our protein. we planned to express them under the Tet regulation.
Regarding the transfection, we are transfecting our gene of interest tagged to mCherry under the Tet regulation.
The problem seems to be in the viruses we have been producing for a while..
Thanks for the information.
A few possibilities on why you are not seeing the signal after transduction:
1. The titer of your lenti is very low therefore very few/if any cells are being infected. Have you titered the lenti?
2. You are not using a high enough MOI. Typically transducing human cells requires an MOI of 5 to achieve >95% transduction efficiency.
3. You are attempting to induce expression with tet too soon after transduction. It takes a few days for the lenti to integrate into the genome. For instance, if you had mCherry under a CMV promotor, you would not see expression until about 4 days post transduction.
Regarding the transfection, since you are transfecting a plasmid you would see the signal much sooner since there is no integration into the genome.
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