Recently we faced an issue where we transduced primary mouse T cells with CAR constructs + a fluorescent tag. We tracked the expression of the tag on day 0, 2, and 4 post-transduction and the tag expression always decreased by ~50% every 2 days. (i.e. We usually have an initial tag expression of 20% which went down to ~5% by day 4). Our negative control (a CAR-negative, tag-only construct) also shows decreases of tag expression similar to the CAR+ constructs.
The initially low transduction efficiency could be due to our producer cells having a low titer (i.e. 1 x10^5 IU/mL) when tested on NIH-3T3. However, I was wondering what could possibly explain the decrease in tag expression over time?
I tried testing different transduction methods (i.e. retronectin, polybrene), MOI, centrifugation speed, cytokine treatments but none seemed to prevent the decreasing tag expression. May I ask if anyone has any suggestions on what other things I can look into to try troubleshooting the issue?
Would also love to hear if anyone has similar experiences working with transducing mouse T cells, such as what would work well and what pitfalls to avoid.
Appreciate any help in advance!
Best,
Oliver
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