Semiquantitative PCR analysis of NOD/SCID bone marrow cells reveals multiple vector copies per transduced cell.

Figure shows agarose gel used to quantify the amount of human DNA (human β-actin) and vector copy number (GFP) per total bone marrow sample. The standard curve for amount of human genomic DNA per sample was generated by mixing mouse and human MNCs at varying dilutions followed by DNA extraction. The well labeled 100 contains 100% human cells followed by 43% human cells and so on. The vector copy standard curve was generated by dilution of DNA from a clonally expanded HeLa cell containing a single EF-1α SIN vector integrant (wells a-d correspond to dilutions containing 300, 100, 30, and 10 pg/μL, respectively, and wells e-k correspond to dilutions of 1500, 900, 300, 100, 30, 10, and 3.3 pg/μL, respectively. The upper panel shows PCR for human β-actin and GFP on mouse bone marrow from cells transduced using the PGK lentiviral vector (mouse nos. 1-3) and EF-1α SIN lentiviral vector (mouse nos. 11-14). The lower panel shows the analysis of bone marrow sample from control mouse 7.23. The copy number values determined from the semiquantitative PCR analysis are shown in Table 1.