3 replies to this topic

[Andrew]

I want to transduce human Tcells with a Foxp3 shRNA.  I've heard that Tcells are very hard to transduce, does anyone have any tips?

Edited by Andrew, 03 November 2009 - 08:40 PM.

[elpollodiablo]

Andrew, on Nov 3 2009, 09:39 PM, said:

I want to transduce human Tcells with a Foxp3 shRNA.  I've heard that Tcells are very hard to transduce, does anyone have any tips?

I'd guess you could do it with a HIV-based lentiviral vector system, as it is already adapted to transducing non-dividing T-cells.

[miBunny]

what vector backbone are you using?  (lentivector or onco-retrovector)?

With activated T cells, I have gotten up to 70% transduction efficiencies with retrovirus infections.  60 minutes spinfection with 1 million cells and 1 mL viral sup.  8 ug/mL of polybrene.

[Functional Screens]

I transduced 1-2 X 10^5 cells/well in 6-well plate with 1-2 ml lentivirus, spinoculation (900-1000 X g for 60min at room temp.) with 8 ug/ml polybrene, change medium after centrifugation.

Edited by Functional Screens, 05 November 2009 - 06:17 PM.