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Keywords:

  • Hematopoiesis;
  • Stem cells;
  • Retrovirus;
  • Integration;
  • PCR

Abstract: The semirandom location of retroviral integration in the target cell genome introduces a marker in the form of a fusion sequence composed of a genomic and a proviral part that is unique for each transduced cell and its clonal progeny. High-sensitivity detection of these fusion sequences would allow the tracking of clonal contributions of individual, marked hematopoietic progenitor, and stem cells in vivo. Clone detection by Southern blot has helped to analyze models of oligoclonal repopulation but is limited in sensitivity and specificity. Inverse PCR (Nolta et al., Proc. Natl. Acad. Sci. USA 93: 2414–2419) can demonstrate the clonal identity by sequencing but does not permit simultaneous detection of multiple clones. In an efficiently transduced rhesus macaque model (Tisdale et al., Blood 92: 2681–2687; Wu et al., Mol. Ther. 1: 285–293) Kim et al. (Blood 96: 1–8) have identified more than 40 insertion sequences from marrow CFU by inverse PCR. However, no previous study has been able to directly analyze the number of clones active in vivo. Here we dem-onstrate that the application of a recently developed PCR technology allows the simultaneous visualization of multiple integration sites from small clonal con-tributions to hematopoietic cells. By combining solid-phase primer extension with ligation-mediated PCR, direct genomic sequencing of retroviral integra-tion sites was obtained in murine bone marrow samples. Further development of this technology will allow analysis of the clonal composition of marked he-matopoiesis in small and large animals as well as in human gene transfer.