Fig. 2

Generation of HLA-exchanged iPSCs. A Schematic diagram of the experimental protocol for generation of HLA-exchanged iPSCs via B2M KO and HLA gene transfer. B2M-sgRNA and target sequence are shown in the middle; construction of the CSII-EF-B2M-A11 vector is shown at the bottom. P1 and P2 arrows were primers for identification of HLA gene transfer. B B2M targeting of C55 iPSCs by CAS9 gene editing. Non-transfected C55 cells (left panel) were used as HLA-I positive control. The non-transfected (left panel) and transfected (middle panel) cells were stained with anti-HLA-ABC-PE antibody and sorted for HLA-I negative (right panel). C HLA gene transfer. Fluorescent images of C55-B2M^KO cells transducted with CSII-EF-hB2M-A11 vector (right panel) or PBS (left panel). Scale bars, 100 µm. D FCM analysis of HLA-I expression. C55-B2M^KO cells transducted by CSII-EF-hB2M-A11 vector was analyzed by FCM with anti-HLA-ABC-PE staining. The Venus/HLA-I double-positive cells were regarded as the successfully transducted cell population. E Identification of HLA gene transfer with specific primers for the CSII-EF-hB2M-A11 vector. The PCR product is approximately 1.3 kb in size. Deionized water was used as the blank control (H₂O) and the CSII-EF-hB2M-A11 vector was used as a positive control (Pos). F Karyotype analysis of the C55-A11#3 line. G-banding of chromosome shows normal karyotype after several rounds of genome manipulations. G H&E staining of sections of teratomas generated from C55-A11 in NCG mice. Three typical tissue types from three germ layers are shown. Scale bars, 20 µm