title: A defined window for efficient gene marking of severe combined immunodeficient-repopulating cells using a gibbon ape leukemia virus-pseudotyped retroviral vector.
abstract: We have investigated the minimal time required for efficient transduction of human hematopoietic repopulating cells using a surrogate nonobese diabetic (NOD)/severe combined immunodeficient (SCID) xenoengraftment assay. Cord blood CD34+ cells were transduced to high levels over 24-48 hr in the presence of Flt-3 ligand, stem cell factor, interleukin 3, and interleukin 6. Under these conditions, high levels of NOD/SCID repopulating activity were preserved, but the levels of gene marking in engrafting cell populations measured by expression of a reporter transgene were low. Extension of the transduction period by 24 hr (total culture period, 72 hr) under the same cytokine conditions resulted in high levels of gene marking, but on closer analysis expression was limited predominantly to the myeloid population. Efficient transduction of both lymphoid and myeloid lineages could be achieved only if the transduction protocol was extended by a further 24 hr (total culture period, 96 hr), suggesting that myeloid lineage-committed precursors are capable of repopulation, and that over shorter time periods transduction is largely restricted to this population. This adds to the emerging evidence of heterogeneity within the SRC compartment, and has important implications for the interpretation of this assay in stem cell transplantation and gene transfer studies.
authors.affiliation: Molecular Immunology Unit, Institute of Child Health, London, United Kingdom.
authors.forename: C
authors.initials: C
authors.lastname: Demaison
authors.forename: G
authors.initials: G
authors.lastname: Brouns
authors.forename: M P
authors.initials: MP
authors.lastname: Blundell
authors.forename: J P
authors.initials: JP
authors.lastname: Goldman
authors.forename: R J
authors.initials: RJ
authors.lastname: Levinsky
authors.forename: M
authors.initials: M
authors.lastname: Grez
authors.forename: C
authors.initials: C
authors.lastname: Kinnon
authors.forename: A J
authors.initials: AJ
authors.lastname: Thrasher
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000235:genetics; D016207:Cytokines / Q000627:therapeutic use; D005434:Flow Cytometry; D005819:Genetic Markers; D005822:Genetic Vectors; D006801:Humans; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D016511:Severe Combined Immunodeficiency / Q000235:genetics / Q000276:immunology* / Q000628:therapy
chemical_list: D018952:Antigens, CD34; D016207:Cytokines; D005819:Genetic Markers