Analysis of the HOXA transforming potential in primary hematopoietic cells. (A) Schematic map of the retroviral constructs used in this study. The expression of the HA-tagged HOXA cDNAs is driven by the long terminal repeat (LTR) promoter of pMSCVhygro (Ψ-packaging signal), whereas the hygromycin resistance gene is under control of a phosphoglycerate kinase (pgk) promoter. (B) HA-specific immunoblot of cellular proteins extracted from retroviral packaging cell lines transfected as indicated. To exclude a potential spill-over of HA-HOXA11 into the HA-HOXA13 lane, expression of HA-HOXA13 is shown again next to a vector control in a separate blot. Please note that because of the highly acidic carboxy-terminus, HA-HOXA7 migrates aberrantly slow in sodium dodecyl sulfate–polyacrylamide gel electrophoresis. (C) Serial replating assays of HOXA-transduced cells. The figure shows colonies arising after 3 replating rounds. Cells were transduced with HOXA genes either individually or in combination with Meis1 as indicated. Two typical examples for each construct are shown of up to 4 experiments performed in total. (D) Replating assays with Meis1. Transduction of Meis1 alone did not have any effect on hematopoietic differentiation, as evidenced by a lack of colony-forming capacity in replating assays like those described previously. (E) Generation of HOXA-transformed cell lines. HOXA or HOXA/Meis1-transduced cells that were able to generate permanently growing cell lines (> 2 months continuously in culture) at least twice in 3 attempts are marked by filled squares. HOXA7 yielded lines only in the presence of Meis1.