A defined window for efficient gene marking of severe combined immunodeficient-repopulating cells using a gibbon ape leukemia virus-pseudotyped retroviral vector

We have investigated the minimal time required for efficient transduction d f human hematopoietic repopulating cells using a surrogate nonobese diabeti c (NOD)/severe combined immunodeficient (SCID) xenoengraftment assay. Cord blood CD34(+) cells were transduced to high levels over 24-48 hr in the pre sence 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 me asured by expression of a reporter transgene were low. Extension of the tra nsduction period by 24 hr (total culture period, 72 hr) under the same cyto kine conditions resulted in high levels of gene marking, but on closer anal ysis expression was limited predominantly to the myeloid population. Effici ent transduction of both lymphoid and myeloid lineages could be achieved on ly if the transduction protocol was extended by a further 24 hr (total cult ure period, 96 hr), suggesting that myeloid lineage-committed precursors ar e capable of repopulation, and that over shorter time periods transduction is largely restricted to this population. This adds to the emerging evidenc e of heterogeneity within the SRC compartment, and has important implicatio ns for the interpretation of this assay in stem cell transplantation and ge ne transfer studies.