Insertional oncogenesis has become the major safety concern for clinical gene transfer into human hematopoietic stem cells, because random retroviral vector integration may activate cellular proto-oncogenes and lead to the emergence of hyperproliferative clones. One potential approach to prevent adverse events while retaining the clinical benefits is to include a suicide gene in the retroviral vector carrying the therapeutic gene; if needed, vector-driven proliferation could be eliminated by drug treatment. Our lab has developed a novel model for experimental insertional oncogenesis in mice to assess the effectiveness of suicide gene therapy. Delivery of a bicistronic MoMLV-based retroviral vector encoding the murine cellular oncogene c-kitD814V to the IL-3 dependent murine leukemia cell line Ba/F3 resulted in vector-driven autonomous cell proliferation after IL-3 withdrawal from the culture medium. The vector was designed to simultaneously express a suicide gene from an internal ribosome entry site (IRES). We tested three different suicide gene candidates: the wild type Herpes simplex virus thymidine kinase (HSV-TK); sr39TK, a mutant created by semi-random mutagenesis that has been reported to have a much higher affinity for Ganciclovir (GCV); and superTK, based on the sr39TK sequence with two silent point mutations introduced to eliminate cryptic RNA splice acceptor and donor site. We derived clonal cell lines from transduced Ba/F3 cells expressing c-kitD814V and either one of the three suicide genes. All cell lines were sensitive to GCV -induced cell death in vitro, and the Ba/F-sr39TK and Ba/F-superTK cells responded to GCV dosages approximately 10 fold lower than the Ba/F-HSV-TK cells. We also characterized GCV-sensitivity of heterogenous, non-selected transduced cell pools to account for suicide escape mutants that may develop due to partial vector loss, splicing events, gene rearrangements or epigenetic silencing. GCV treatment for 21 days of Ba/F3 cell pools transduced with HSV-TK or sr39TK resulted in emergence of GCV resistance; in contrast, proliferation was consistently inhibited by long-term GCV treatment of Ba/F3 cells transduced with superTK. The cell lines transduced to express c-kitD814V were leukemogenic in vivo when injected i.v. into immunocompetent, syngeneic Balb/c mice. Moreover, leukemogenicity was strictly dependent on vector insertion since mice challenged with non-transduced Ba/F3 cells did not develop leukemia. GCV treatment (50 mg/kg i.p.) of Balb/c on five consecutive days one week after challenge with 5,000 Ba/F-HSV-TK cells did not protect from leukemia development, and the mice succumbed to disease around day 21 post-challenge. In contrast, the same GCV treatment of mice injected with 5,000 Ba/F-sr39TK or Ba/F-superTK led to 100% survival 40 days post-challenge. Thus, in this model of vector-driven insertional oncogenesis, a suicide gene approach was effective for eliminating leukemia using modified HSV-TK variants with improved biological activity and diminished aberrant splicing.