We want to transduce T-and B-lymphocyte cell lines but transduction efficiencies are poor, i.e. western blots are negative but RT-PCR is positive.
Is your protein stable? You might already have done that, but I would include something like GFP or other easy gene to differentiate between actual transduction efficiency and secondary expression problems.
I used this system and felt that the expression driven by CMV promoter was very poor in B and T cells as almost no GFP positive cells could be seen.
Dear Gerco, thank you very much for your response!
We did transduce pLenti6.3/GFP into B- and T-cell lines but only 3% showed GFP positivity (FACS-Analysis).
The Tetracycline Repressor (TR) could be transduced quite efficient into HT1080 (Western blot positive) but we failed to detect the TR in T- or B-cell lines following transduction.
I'm very happy that there is somebody out there, like you, to give suggestions or help to solve the problems. Do you actually work with this lentiviral system?
Looking forward to hearing from you.
Kind regards, Burkhard
No, years ago I worked, with the gateway system, and setup things for actually using lentiviral system fiddling around with hybridoma's but I actually wonder if in the end I used it, or that an alternative gave quicker results. But if even GFP is expressed poorly, this must mean that indeed either the promotor is not ideal or more likely you hit indeed too little. I looked quickly to my protocols, I only have protocol for adherent cells wich is probably not much of a help.
Thanx to Jia-Wang, thanx Gerco!
Unfortunately, T- as well B-cell lines seem to be transduced only marginal since transduction of HT1080 worked out quite good.
I do not know if I should change to a different lentiviral expression vector that carries WPRE, like pRRL, or if I could increase transduction efficiencies somehow?
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