Southern blot analysis of BM samples from mice transplanted with transduced cord blood cells.

Genomic DNA from BM cells harvested at 90 to 120 days after transplantation was digested with BamHJ or Asp718, and hybridized with probes for human CD4 and EGFP. (Panel A) Representative analysis of a human CD4 hybridization of BM DNA digested with BamHJ. Quantification of engraftment was done by densitometric analysis of test and control DNA samples prepared by mixing graded proportions of human and mouse DNA. Mouse 4L was transplanted with purified EGFP⁺ cells, and mice 2G and 2H with purified EGFP⁻ cells. The correlation analysis of these data with respect to CD45⁺determinations (Tables 2 and 3) is shown in the plot. (Panel B) EGFP hybridizations of BM DNA samples digested with Asp 718 or BamHJ. A standard curve of the number of proviral copies per cell was constructed by mixing different proportions of DNA from a cell line bearing a single copy of EGFP with DNA from nontransduced cells. The correlation analysis of the estimated number of copies per BM cell with respect to determinations of the proportion of CD45⁺/EGFP⁺ cells in total BM (Tables 2 and 3) is shown in the plot. To investigate the clonal composition of the engrafted transduced human cells, samples were digested with BamHJ. Restriction sites within the EGFP1 vector are also shown.