Figure 1

Generation of conditionally immortalised myeloid progenitor cell lines co-expressing Hoxa9 and Meis1 in the presence of doxycycline. (a) Schematic representation of multimodal retroviral vector and integrated provirus to enable constitutive Hoxa9.EGFP and inducible Meis1.dTomato gene co-expression. RSV, Rous sarcoma virus promoter; Δ, SIN configuration with partially deleted U3 of the 3′ long terminal repeat (LTR); ψ, packaging signal; PRE, post-transcriptional regulatory element; EGFP, dTomato, fluorescent proteins. (b) Experimental design. Murine Rosa26rtTA lineage negative cells were transduced on day 1 under myelomonocytic conditions (36S) without doxycycline (DOX). On the next day, cells were split into DOX untreated (DOX-, OFF) and DOX treated cultures, which were supplemented with 1 µg/mL DOX (DOX+ , ON). HOXA9.EGFP⁺/MEIS1.dTomato⁺ double-positive cells (DOX+) and HOXA9.EGFP⁺ (DOX-) were sorted on days 13–23 after transduction followed by a limiting dilution assay to select stable H9M-ciMPs. (c) The number of potential cell lines for DOX+ and DOX- conditions (~ day 45 after transduction). The experiment was performed independently for multimodal retroviral vectors with (MRV) or without (MRV*) LM-PCR linker in three biological replicates for DOX+ and two biological replicates for DOX- conditions (one biological replicate / one 96-well plate). (d-e) The number of generated, pre-selected for LM-PCR analysis and selected cell lines for MRV and MRV* vectors under DOX supplementation. (f) Average vector copy number (VCN) determined for pre-selected (n = 10 for MRV, n = 12 for MRV*) H9M-ciMPs. (g) Gene expression levels of EGFP⁺dTomato⁺ double-positive cells in selected (n = 8) H9M-ciMPs after cryopreservation (pre-cultured ~ 3 months in 36S conditions before cryopreservation). Horizontal lines represent the mean values. Each data point represents the result of an individual cell line. LM-PCR, ligation-mediated PCR. H9M-ciMPs, conditionally immortalised myeloid progenitor cell lines engineered to co-express Hoxa9 and Meis1. (d–g) generated in Prism 5 (GraphPad software, San Diego, CA).