Humanized BLT Mouse Construction

NSG (NOD/SCID/IL2rγ −/−) mice were used to generate humanized BLT mice and housed according to UCLA Humanized Mouse Core Laboratory procedures as previously described. ³¹ Human fetal thymus and fetal liver were obtained from Advanced Bioscience Resources (ABR). Fetal tissues were obtained without patient identifying information. Written informed consent was obtained from patients for the use of tissues for research purposes. Briefly, one day before transplant, CD34+ cells were isolated from fetal livers using anti-CD34+ magnetic bead-conjugated monoclonal antibodies (Miltenyi Biotec) and transduced with vectors described above. NSG mice were conditioned with sub-lethal body irradiation (270 cGy Cesium-137). On the day of transplant, an equal mixture of non-transduced or vector-transduced FL-CD34+ cells (~ 0.5×10⁶ per mouse) and CD34- cells (~4.5 ×10⁶ per mouse) were mixed with 5μL of Matrigel (BD Biosciences) and implanted with a piece of thymus under the kidney capsule. Mice were then injected with non-transduced or vector-transduced CD34+ HSPCs (~ 0.5×10⁶ per mouse) using a 27-gauge needle through the retro-orbital vein plexus. At 8–10 weeks post-transplantation, blood was obtained from each mouse by retro-orbital sampling and peripheral blood mononuclear cells were analyzed by flow cytometry to quantify human immune cell engraftment.