Serial transplantations in NOD/SCID mice.

(Panel A) FACS profile of marrow cells from a representative NOD/SCID mouse that 8 weeks earlier had received a transplant of 2 × 10⁵ infected CB CD34⁺ cells that had been expanded for 4 weeks. The BM of the primary mouse was injected into a secondary sublethally irradiated NOD/SCID mouse; the BM of this mouse was injected into a tertiary mouse. FACS analysis of human CD45 expression in the BM of primary, secondary, and tertiary mice was performed on total BM cells. The numbers in the top right quadrants show the percentages of GFP⁺ cells within the CD45⁺ population (panel B) FACS histograms representing human GFP⁺ cells on total BM population of the same primary, secondary, and tertiary recipients shown in panel A. (Panel C) GFP transgene amplification curves, obtained by real-time quantitative PCR analysis, of 100 ng DNA from the same primary, secondary, and tertiary mice shown in panels A and B (shown in Table 3 as mice A₅, A_5.1, and A_5.1.1). A standard curve was obtained by using increasing amounts of vector plasmid as follows: 0.0086 ng (Ct: 26.1), 0.086 ng (Ct: 22.9), 0.86 ng (Ct: 19.5), 8.6 ng (Ct: 16.4), and 86 ng (Ct: 14.2). To calculate the vector copy number per genome (human plus murine) we used HeLa cell clone (C3) containing one copy of GFP vector, as previously assessed by Southern blot analyses. The amplification curve obtained with a 10-fold dilution of C3 DNA, corresponding to 0.1 copy of vector per genome, is shown (Ct: 25.5). The analyses indicate that the BM of primary (Ct: 25), secondary (Ct: 27), and tertiary (Ct: 28.22) mice contained 0.10 (corresponding to 10% of the total cells containing one copy number of the transgene, in agreement with the FACS analysis shown in panel A), 0.02 (corresponding to 2% of the total cells containing one copy number of the transgene, in agreement with the FACS analysis shown in panel A), and 0.008 (corresponding to 0.8% of the total cells containing one copy number of the transgene, in agreement with the FACS analysis shown in panel A) copies of vector per genome (human plus murine), respectively. The corresponding Southern blot analysis for human engraftment is shown in Figure 5 (sample A). One of 2 analyses giving similar results is shown. NT indicates untreated mouse; Ct, the cycle number at which the fluorescence signal was more than 10 SD of the mean background noise collected from the 3rd to the 15th cycle.