Mice with targeted genetic alterations are the most effective tools for deciphering organismal gene function. We generated an ENU-based parallel C3HeB/FeJ sperm and DNA archive characterized by a high probability to identify allelic variants of target genes as well as high efficiencies in allele retrieval and model revitalization. Our archive size of over 17,000 samples contains approximately 340,000 independent alleles (20 functional mutations per individual sample). Based on an estimated number of approximately 30,000 mouse genes, the parallel sperm/DNA archive should permit the identification and recovery of ten or more alleles per average target gene which translates to a calculated 99% success rate in the discovery of five allelic variants for any given average gene. The low rate of unrelated ENU-induced passenger mutations has no practical impact on the analysis of the allele-specific phenotype at the G3 generation because of dilution and free segregation of such unrelated passenger mutations. To date 39 mouse models representing 33 different genes have been recovered from our archive using in vitro fertilization techniques. The generation time for a murine model heterozygous for a mutation in a gene of interest is less than 2 months, i.e., three to four times faster compared with current embryonic stem-cell-based technologies. We conclude that ENU-based targeted mutagenesis is a powerful tool for the fast and high-throughput production of murine gene-specific models for biomedical research.
We examined the role of total body magnetic resonance imaging (TB-MRI)-governed involved compartment irradiation (ICI) and high-dose chemotherapy (HDC), followed by stem cell rescue (SCR) in patients with high-risk Ewing tumors (ETs) with multiple primary bone metastases (high-risk ET-MBM). Eleven patients with high-risk ET-MBM receiving initial assessment of involved bones by TB-MRI were registered from 1995 to 2000 (group A). In all, 6 patients out of 11 had additional lung disease at initial diagnosis; all had multifocal bone disease with more than three bones involved. After systemic induction with etoposide, vincristine, adriamycin (doxorubicin), ifosfamide, and actinomycin D (EVAIA) or VAIA chemotherapy, ICI of all sites positive by TB-MRI was administered, followed by HDC and SCR. A second group matched for observation period and consisting of 26 patients with more than three involved bones at diagnosis was treated with the European Intergroup Cooperative Ewing Sarcoma Study-92 (EICESS-92) protocol (group B). These patients did not receive TB-MRI and consequently did not receive TB-MRI-governed ICI, or HDC and SCR. Survival in group A vs group B was 45 vs 8% at 5 years and 27 vs 8% at 10 years after diagnosis (log rank and Breslow: Po0.005). We conclude that TB-MRIgoverned ICI followed by HDC and SCR in ET-MBM is feasible and warrants further evaluation in prospective studies.
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