Figure 4

The simHDR based direct cloning of HLA-A genomic region. (a) Schematic representation of the simHDR illustrating the loading of genomic DNA sequence from human cells onto the 10MAC2. Arrows indicate the position of PCR primers used for analysis. (b) Preparation of PCR HDR donor fragments. Confirmation of precise amplification by electrophoresis. For gel source data, refer to Supplementary Fig. S15. (c) Image of DT40 cells carrying the 10NAC2-HLA-A. EGFP expression indicates the presence of the 10NAC2-HLA-A. BF, bright field. Scale bar: 100 µm. (d) Representative image of metaphase FISH analysis with mouse Cot-I (red) detecting 10MAC2 and HLA-A or 5’EGFP and 3’EGFP-BS PCR HDR donor fragments (green). Arrowhead indicates the 10MAC2 and the inset shows an enlarged image thereof. Scale bar: 10 μm. (e) RT-PCR products generated from DT40-10MAC2-HLA-A and DT40-10MAC2 cells cDNAs. Each end point PCR products are shown. For gel source data, refer to Supplementary Fig. S16. (f) Detecting HLA-A protein by western blotting. For membrane source data, refer to Supplementary Fig. S17.