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,
Iīm 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 wonīt 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 donīt 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 donīt work...I transfect gag/pol + vsv + my construct in 293T cells ang get 2x105 TU/ml.....
Regards,
Leo Chicaybam