Figure 2.

Stable Expression of GFP following Integration into the T Cell Genome by HDR

(A) Diagrams of AAV vectors used to transduce cells. AAV:GFP is a vector enabling transient expression of GFP in transduced cells driven by a cytomegalovirus (CMV) promoter. AAV:TRAC:GFP contains a GFP transgene with expression driven by the JeT promoter and flanked by homology arms on the 5′ (L TRAC HA) and 3′ (R TRAC HA) sides to enable targeted integration. (B and C) T cells were mock-electroporated or electroporated with TRC1-2 mRNA and then mock transduced (No AAV) (B) or immediately transduced with AAV:GFP or AAV:TRAC:GFP at an MOI of 1e5 vector genomes (vg)/cell (C). Cells were cultured with 10 ng/mL IL-2 until 7 days post-electroporation, at which point they were cultured in medium containing 10 ng/mL IL-7 and 10 ng/mL IL-15 for the duration of the experiment. Flow cytometry was used to evaluate GFP expression 3, 11, and 21 days post-transduction.