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

Retroviral vector-medial ed expression of the homeoboxgene, HoxB4, in hemat opoietic cells has been reported to mediate a benign expansion of gene-modi fied hematopoietic stem and precursor cells in vivo. In the present study, we used a novel coexpression strategy for coordinated expression of HoxB4 a long with a cytoplasmic protein from a retroviral vector. The novel coexpre ssion strategy, based on cotranslational protein separation mediated by the 2A sequence of foot-and-mouth disease virus (FMDV), allows an indirect qua ntification of HoxB4 expression levels when inserting a reporter such as th e enhanced green fluorescent protein (GFP) in the retroviral vector. Presen ce of the 2A sequence did not interfere with the correct subcellular locali zation of HoxB4 (nuclear) and GFP (cytoplasmic), nor with the titer of bici stronic vectors, and mediated functional long-term coexpression (at least 1 year) of GFP and HoxB4 after transplantation of transduced mouse bone marr ow cells in mice.