Downregulation and loss of proviral expression have been demonstrated to occur in a variety of in vitro studies and in mouse models. Here we evaluated the kinetics of proviral expression after transplantation in a competitive repopulating model in the baboon. Transgene persistence and green fluorescent protein (GFP) expression in peripheral blood leukocytes (PBL) were analyzed in four animals by semiquantitative PCR and flow cytometry for up to 80 weeks (range 17-80). All animals were transplanted with cells transduced with EGFP or EYFP reporters driven by Moloney murine leukemia virus (MoMuLV) or a modified promoter/enhancer, (MND) respectively. Simultaneous dual-color analysis of fluorescence levels in granulocyte and lymphocyte subsets following hematopoietic reconstitution demonstrated progressive loss of fluorescence intensity occurring predominantly early after transplant in cells transduced with both retrovirus backbones and at serial time points. In addition, we carried out PCR analysis of DNA extracted from sorted EGFP-/EYFP- cells and confirmed the presence of cells genetically marked by either vector in this population, indicating the persistence of cells that have downregulated or lost retroviral gene expression. In comparison to mouse studies, however, we did not detect substantial differences between MND and MoMuLV backbones.
- Dual-color fluorescence analysis
- Green fluorescent protein
ASJC Scopus subject areas
- Molecular Biology