title: A model for the detection of clonality in marked hematopoietic stem cells.
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 demonstrate that the application of a recently developed PCR technology allows the simultaneous visualization of multiple integration sites from small clonal contributions to hematopoietic cells. By combining solid-phase primer extension with ligation-mediated PCR, direct genomic sequencing of retroviral integration sites was obtained in murine bone marrow samples. Further development of this technology will allow analysis of the clonal composition of marked hematopoiesis in small and large animals as well as in human gene transfer.
authors.affiliation: Department I of Internal Medicine, University of Freiburg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Schmidt
authors.forename: H
authors.initials: H
authors.lastname: Glimm
authors.forename: N
authors.initials: N
authors.lastname: Lemke
authors.forename: A
authors.initials: A
authors.lastname: Muessig
authors.forename: C
authors.initials: C
authors.lastname: Speckmann
authors.forename: S
authors.initials: S
authors.lastname: Haas
authors.forename: P
authors.initials: P
authors.lastname: Zickler
authors.forename: G
authors.initials: G
authors.lastname: Hoffmann
authors.forename: C
authors.initials: C
authors.lastname: Von Kalle
mesh_terms: D000818:Animals; D001773:Blood Cells / Q000166:cytology; D015139:Blotting, Southern; D001854:Bone Marrow Cells / Q000737:chemistry / Q000166:cytology; D002999:Clone Cells / Q000737:chemistry / Q000166:cytology; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis*; D005819:Genetic Markers; D006085:Graft Survival; D006410:Hematopoiesis; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000166:cytology*; D009052:Leukemia Virus, Murine / Q000235:genetics* / Q000302:isolation & purification; D051379:Mice; D008954:Models, Biological; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics*; D012680:Sensitivity and Specificity; D016662:Virus Integration / Q000235:genetics*
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D005819:Genetic Markers