Targeted GFP expression in MHC II⁺ human cells before and after NOD/SCID mouse engraftment with DR.GFP-transduced CD34⁺ cells.

(A) Analysis of GFP and CD34 expression in human CB CD34⁺cells 3 days after DR.GFP, EF.GFP, or mock transduction. (B) Differentiation of the transduced CB CD34⁺ cells in methylcellulose for CFC assays. Representative photographs of BFU-E, CFU-GM, and CFU-mix under light (top panels) and fluorescence (bottom panels) microscopy after 14 days in culture by an inverted microscope (magnification, × 50). The percentages of GFP bright (GFP⁺) colonies in each group were calculated, and the averages of triplicates are shown at the bottom. (C) FACS analysis of engrafted human cells in the NOD/SCID BM 10 weeks after transplantation with DR.GFP- or EF.GFP-transduced CB CD34⁺cells (mice no. 15 and 20, respectively, in Table 1). Engrafted human cells were first identified in dot plots (top panels, human CD45 versus forward scatter) and gated for further analysis. The percentages of human (CD45⁺, shown in boxes) cells are indicated at the top of dot plots. Further examinations of GFP expression versus human CD34, CD19, CD13, and MHC II markers in gated human CD45⁺ populations are illustrated (bottom panels). (D) FACS analysis of GFP and MHC II expression in gated human populations in the spleen of engrafted NOD/SCID mice. (E) Quantitative analysis of transgene expression specificity in human MHC II⁺ populations found in the NOD/SCID BM and spleen (SP) after engraftment of DR.GFP- or EF.GFP- transduced CD34⁺ cells.