7 replies to this topic

[Shiny]

Hello All,

I am really confused looking at the FACS readout of my transduced primary mouse T cells. I hope somebody might be of help and shed some intellectual insight on the problem.

I used PlatE packaging cells lines and a retroviral plasmid construct (which has 5'LTR promoter, gene of interest tagged to YFP, IRES and HuCD2) to produce virus particles which was used to transduce T cells. I also infected 3T3 cells with the same virus.

The FACS readout shows a good expression of HuCD2 (detected with PE) and also YFP for the 3T3 cells. However when in T cells I just have the expression of HuCD2 whereas I do not have a signal for YFP.

What could be the problem? I have checked the compensation on Caliber that does not seem to be the issue.

Does anybody know if LTR is not suitable as promoter in a T cell.

I am at complete loss to understand the results.

Any help/ ideas is deeply appreciated.

thanks

Shiny

[Rsm]

Have you tried activating the cells? Most viral promoter are off in quiescent T cells (HIV latency for example). But then you also shouldn't see HuCD2...

[Shiny]

Yes I activated the T cells, they don't get transduced when they arent activated.

[Rsm]

Maybe you have degradation of your fusion protein. How about overexpressing the protein of interest alone? You have IRES-hCD2, that should be good enough as a reporter.

[Shiny]

When you say overexpressing of the protein of interest alone,do you mean to drop out YFP tag? But then how would you be able to say if the protein is expressing because although the Gene of interest and HuCD2 is expressed as a single transcript message, they are translated as two proteins due to the presence of IRES, so how would you be able to say that the protein of interest is there without the presence of a reporter.

[Rsm]

Shiny, on 25 March 2011 - 07:32 AM, said:

When you say overexpressing of the protein of interest alone,do you mean to drop out YFP tag? But then how would you be able to say if the protein is expressing because although the Gene of interest and HuCD2 is expressed as a single transcript message, they are translated as two proteins due to the presence of IRES, so how would you be able to say that the protein of interest is there without the presence of a reporter.

Expression from IRES-reporter is usually considered sufficiently significant to say your GOI is there as well. Fusion however, can severely alter the function (or stability) of your gene. You have the YFP IRES-hCD2 construct working in some cells, right? Why don't you test expression of YFP versus hCD2 in those cells? From my experience, the correlation of IRES is r^2>0.9999 and p around e^-8. If you want, you can also change your construct to a 2A peptide, that is even more significant (p>e^-11, if I remember correctly... I had the data somewhere...).

[Shiny]

how would you test expression of YFP vs hCD2, by FACS.. as in how do you correlate or quantify?

[Rsm]

Now you ask a tricky question...
Wait, I had it somewhere...

I have used this one: http://research.stow...tract/index.htm, with which you can export your .fcs files to excel and calculate the R2 value. I think it is for free (as in beer, not as in speech).