Plenti giii cmv gfp 2a puro

Lentiviral constructs encoding control shRNA-GFP (SHC016V0718127MN) or ZFYVE21 shRNA-GFP (TRCN0000436194) constructs were purchased from Sigma-Aldrich. Lentiviral constructs encoding ZFYVE21-GFP (pLenti-GIII-CMV-GFP-2A-Puro) were purchased from Applied Biological Materials. CD4⁺CD45RO⁺ T cells (5×10⁴ cells per well) were electroporated with 2μg of the plasmids above according to the manufacturer’s specifications (Amaxa, #VPA-1002) using the Nucleofector II device (Amaxa). Seventy-two hours later, for a total of 4 d in culture, T cells were washed and restimulated with plate-bound anti-CD3 (1μg/mL) and treated as indicated in the text. Lentivirus containing the constructs above were used to infect 1×10⁵ human CD34+ stem cells for 8 hrs. The next day, transduced GFP⁺ stem cells were FACS-sorted and intrahepatically injected into sublethally irradiated (180 cGy) 1–3dy old female NSG (Jackson, #00557) pups. Nine-to-twelve weeks later, mice were bled and human Tmem were analyzed as indicated in the text. In certain experiments, 8×10⁶ PBMCs from the mice above were passively transferred i.p. into adult 6–12-week-old SCID/bg hosts containing human artery segments implanted into the infrarenal position of the descending aorta. Four weeks later, grafts and host sera were harvested for analysis.