Retroviral vector-mediated expression of HoxB4 in hematopoietic cells using a novel coexpression strategy

Retroviral vector-medial ed expression of the homeoboxgene, HoxB4, in hematopoietic cells has been reported to mediate a benign expansion of gene-modified hematopoietic stem and precursor cells in vivo. In the present study, we used a novel coexpression strategy for coordinated expression of HoxB4 along with a cytoplasmic protein from a retroviral vector. The novel coexpression strategy, based on cotranslational protein separation mediated by the 2A sequence of foot-and-mouth disease virus (FMDV), allows an indirect quantification of HoxB4 expression levels when inserting a reporter such as the enhanced green fluorescent protein (GFP) in the retroviral vector. Presence of the 2A sequence did not interfere with the correct subcellular localization of HoxB4 (nuclear) and GFP (cytoplasmic), nor with the titer of bicistronic vectors, and mediated functional long-term coexpression (at least 1 year) of GFP and HoxB4 after transplantation of transduced mouse bone marrow cells in mice.