DNA analysis of vector proviruses.

(A) BM colonies grown in soft agar were individually picked and subjected to PCR with primers that amplify a 403-bp fragment of foamy virus vector DNA. Representative results from 27 colonies obtained at 6 months after transplantation are shown, 22 of which contained the vector genome. Negative controls included water and DNA from an untransduced BM colony. (B) Genomic DNA samples from BM and spleen (SP) of long-term and secondary transplant recipients (indicated by number) were digested with EcoN I and probed for vector sequences (Figure 1 has locations). Standards were untransduced mouse spleen DNA mixed with DNA from diploid fibroblasts containing a single integrated HFV vector provirus (9.8-kb EcoN I fragment) to generate the indicated copy numbers. The upper blot was probed with HFV sequences. To correct for sample loading, the same blot was reprobed with mouse genomic β-glucuronidase sequences to detect a 3.8-kb fragment (GUS probe). The calculated copy numbers based on this PhosphorImager analysis are shown below each lane (provirus copies per diploid genome). (C) Integration site analysis using Nsi I to assay for unique-sized junction fragments. Positive and negative control samples were 100% and 0% standards of panel B, respectively. The positions of size standards are indicated in each panel.