Hello everyone, we are trying to transduce T-lymphocytes (TLs) with vector containing IRES, and cannot see any expression using flow cytometry (our target protein is conjugated with GFP). The problem is that the "basic" vector that does NOT contain IRES is well expressed in TLs. Therefore we suppose that either vector is "bad" or TLs has limitations of some sort that I do not know (smth like IRES is not very "welcome" in TLs - whatever it may be)... I hope that I am mistaken (there's no clear answer for that in PubMed for ex.) but if someone has more deep experience on that matter - would be very much obliged for the help.
Thanking in advance!
Hello Syed.
Thank you for the answer. I was talking about transDUCTION - when you "infect" your T-cells with a virus that contain your GOI.
The construct is, actually, GOI1-IRES-gfpGOI2. And it works perfectly when we transFECT HeLa cells. Also the construct gfpGOI2 alone works well in T-cells also.
While the bicistronic one with IRES does not work in T-cells...
Maybe it's worth to try to transduce (transDUCTION) Jurkats?
Viruses have a packaging limit and IRES sequences are quite large. You may be approching your vector's limit and, therefore, generating low titer virus. This would possibly explain the difference between the base vector and the IRES one.
What viral system are you using? How big are your GOIs?
Which IRES sequence are you using and have you tested functionality of your IRES in other cells; e.g. 293T cell transfection? We have successfully used the Encephalomyocarditis virus (ECMV) IRES extensively in T-cells. However it has been reported that IRES mediated gene expression is reduced when compared with that from the primary transgene1. We have also noted that the reduction in transgene expression following an IRES appears to correlate with the size of the primary transgene. If your primary transgene is >1kb, you may want to consider viral '2A' sequences as an alternative bicistronic expression strategy.
1. Mizuguchi, H., Xu, Z., Ishii-Watabe, A., Uchida, E. & Hayakawa, T. IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Mol Ther 1, 376-382 (2000).
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