Help: protocols for shRNA knockdown effect on Ag-specific Treg differentiation - (Oct/11/2010 )
Hi, all, I need help for protocols of shRNA knockdown effect on Ag-specific Treg differentiation in vitro and/or in vivo.
Now, I want to check the effect of my shRNA on Ag-specific Treg differentiation both in vitro and in vivo. But I could not find the proper protocols.
1. For in vitro assay, I thought I should first stimulate naive OT-II T cells with OVAp and irradiated APC, then do shRNA included retrovirus infection 2 days after T cell stimulation. But I was told by several persons that this procedure would not work because virus can not reach T cells since APC is there. So it means I have to use anti-CD3 and anti-CD28 instead of APC? I am not sure about it. And I wonder if I can overcome this problem by using puromycin selection after virus infection or not. I need suggestions from experienced experts and thanks a lot in advance!
2. For in vivo assay, I plan to use puromycin selection after retrovirus infection. I just wonder usually after shRNA included retrovirus infection how long I should culture the OT-II T cells with puromycin before doing adoptive transfer. I cannot find enough such information from publications.
If somebody can share his protocols or give any suggestions, I appreciate it very much!
yg_eagle on Mon Oct 11 17:06:06 2010 said:
Hi, all, I need help for protocols of shRNA knockdown effect on Ag-specific Treg differentiation in vitro and/or in vivo.
Now, I want to check the effect of my shRNA on Ag-specific Treg differentiation both in vitro and in vivo. But I could not find the proper protocols.
1. For in vitro assay, I thought I should first stimulate naive OT-II T cells with OVAp and irradiated APC, then do shRNA included retrovirus infection 2 days after T cell stimulation. But I was told by several persons that this procedure would not work because virus can not reach T cells since APC is there. So it means I have to use anti-CD3 and anti-CD28 instead of APC? I am not sure about it. And I wonder if I can overcome this problem by using puromycin selection after virus infection or not. I need suggestions from experienced experts and thanks a lot in advance!
2. For in vivo assay, I plan to use puromycin selection after retrovirus infection. I just wonder usually after shRNA included retrovirus infection how long I should culture the OT-II T cells with puromycin before doing adoptive transfer. I cannot find enough such information from publications.
If somebody can share his protocols or give any suggestions, I appreciate it very much!
Hi,
I´m not an expert, but here you go:
1- I think that you can try to transduce T cells in coculture with APCs. The only problem here is that APCs will compete for the virus binding (but won´t be transduced). To solve this you can increase the MOI, however this can be toxic to T cells. You can use Puro selection, but if the transduction rate is low it will be difficult to maintain the positive cells alive and/or expand them.
anti-CD3/CD28 might be an alternative, but I don´t know how is the Treg differentiation under these conditions. Does it occur? If yes, certainly it would be a cleaner system.
2- I use 0,5ug/ml of Puro to select Jurkat lymphocytes. Three days after I have an almost pure population. Ideally you should make a dose response curve in primary lymphocytes (trypan blue exclusion).
Which cell line/system you use to produce retrovirus? I also work with genetic modification of T cells, but I use lenti. In my hands retrovirus production simply don´t work...I transfect gag/pol + vsv + my construct in 293T cells ang get 2x105 TU/ml.....
Regards,
Leo Chicaybam