Rapid generation of a tetracycline-inducible BCR-ABL defective retrovirus using a single autoregulatory retroviral cassette

The development of chronic myelogenous leukemia (CMIL) models in mice using an inducible BCR-ABL gene has been hampered by the requirement of sequenti al expression of tTA (Tet repressor-VP16 fusion protein) and Tet-OP sequenc es in the same cells after separate transfection. This double transfection strategy is time consuming as it requires screening of many hundreds of ind ividual clones and cannot be applied to primary hematopoietic cells. To gen erate a tetracycline-inducible BCR-ABL retrovirus, we have subcloned BCR-AB L p210 cDNA in the SIN-Retro-TET vector, which allows regulated expression of a gene of interest in a single autoregulatory cassette, containing both tTA and Tet-OP sequences. Retroviral particles were obtained by transfectin g the SIN-BCR-ABL p210 construct into the 293 cells and by VSVG pseudotypin g. To determine the functionality of the retrovirus, the IL-3-dependent mur ine Ba/F3 cell line was retrovirally transduced and clones were grown in th e absence of both IL-3 (to select for transformed cells) and a tetracycline analog, doxycycline (to induce BCR-ABL expression). Using this technique, polyclonal Ba/F3 cells and several growth factor-independent Ba/F3 clones e xpressing BCR-ABL were obtained within 2-3 weeks. A single dose of doxycycl ine added to the medium (1 mug/ml), induced in different clones, a reductio n of BCR-ABL protein levels by 60-90% at 24 h, leading to cell death in the absence of IL-3. In several individual clones, BCR-ABL expression was furt her reduced to become almost undetectable at 48 In. The doxycycline-regulat ed BCR-ABL expression was stable, as many clones maintained in culture for >8 months showed a persistent inhibitory response to doxycycline addition i n the medium. In in vivo experiments, subcutaneous injection of 2 x 10(6) B a/F3-SIN p210 cells in nude mice induced visible tumors in 2 weeks and all established tumors completely regressed upon addition of doxycycline in the drinking water (200 mug/ml). To determine the functionality of the inducib le BCR-ABL retrovirus in vivo, primary Lin(-) bone marrow cells were transd uced with SIN-p210 and transplanted in lethally irradiated mice. All transp lanted mice had successful hematopoietic reconstitution and BCR-ABL integra tion was found in the peripheral blood of seven out of 14 mice available fo r long-term analysis (>6 months). However, despite evidence of retrovirus-m ediated gene transfer, there was no evidence of leukemia, due either to low viral titers or to the relative inefficiency of the minimal CMV promoter i n primary hematopoietic cells. Thus, these results demonstrate for the firs t time, to our knowledge, the feasibility to generate an inducible BCR-ABL retrovirus in a single step, in the context of an immortalized cell line. O ur data suggest that with further improvements of the retrovirus-mediated g ene transfer technology, it might be possible to generate inducible leukemi a models in mice by the use of single retroviral constructs.