Retroviral vectors mediating different levels of HOXB4 expression.

(A) Schematic presentation of MSCV- and FMEV-based retroviral vectors used for HOXB4 expression in this study. The cotranslational separation activity of the 2A-sequence of foot-and-mouth disease virus (FMDV) enables concordant expression of GFP and hemagglutinin epitope–tagged (▪) HOXB4 at constant molar ratios. Pgk indicates phosphoglycerate kinase promotor; neo, neomycin resistance gene; wPRE, posttranscriptional regulatory element of the woodchuck hepatitis virus. (B) Quantification of ectopically expressed HOXB4 protein in K562 cells that were transduced with the retroviral vectors presented in panel A with the use of an MOI of less than 1. The vector MSCV-GFP31 was used as negative control (lane 2). Transduced cells were either selected with the use of G418-containing medium (lane 1) or sorted to greater than 90% GFP⁺ cells (lane 2-5). Crude extracts were separted by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and HAHOXB4 proteins were detected immunologically by means of an anti-HA antibody. The relative levels of HOXB4 expression presented in panel A for each vector were determined by quantifying HAHOXB4-specific signal intensities by scanning densitometry as described in “Materials and methods.” (C) Detection of ectopic HOXB4 expression in human colony-forming cells. Crude extracts of human CFCs, which were derived from the bone marrow of NOD/SCID mice that had received transplants (experiment 2, Tables 1 and2) were treated as described in panel B. HAHOXB4 protein was detected in CFCs derived from mice that had been injected with human FMEV-HAHOXB4wPRE–transduced CD34⁺ cells (lane 3 and 5), but not in progenitors obtained from mice that either had received transplants of RFP-transduced CD34⁺ cells (lane 2) or had simultanously received injections of RFP and GFP control vector–transduced CD34⁺ cells (lane 4). Lane 1 (+): K562 extract corresponding to lane 5 in panel B as a positive control. Note that the GFP2AHAHOXB4 fusion protein (62 kDa; indicated by arrows) that was observed in transduced K562 cells is almost undectable in these primary human cells.