Disease incidences and LOH in established leukemias of Icsbp^−/−Nf1^+/− mice. (A) Allelic imbalance of Nf1 wt-allele expression in established leukemias. RT-PCR with allele specific primers was performed on cDNA-derived from BM of mice with established leukemias to investigate imbalanced expression of wild-type and mutated allele. The top band shows an amplification of a 511-bp fragment from the Nf1-wt allele transcript and the bottom band a 340-bp fragment of the Nf1-neomycin hybrid transcript. Actin expression was used as control. X denotes absence of wild-type band; (X), reduced wild-type band. Control lanes 8 to 12: mice without leukemia. (B) No allelic imbalance is observed in mice without disease manifestation. RT-PCR with allele specific primers was used to show balanced expression of wild-type and mutated Nf1 allele in young (3 months) and old (8-9 months) mice without disease manifestation. (C) Allelic imbalance of Nf1-wt allele expression in leukemias is the result of LOH. PCR with allele specific primers was performed on genomic DNA isolated from BM of diseased mice. In all but one case where an imbalanced allelic expression was observed, the wild-type band was lost or reduced in intensity. X indicates absence of wild-type band; (X), reduced wild-type band. (D) Replication of LOH in transplanted NOD/SCID mice. PCR with allele-specific primers was performed on genomic DNA of established leukemias isolated from BM of diseased NOD/SCID mice transplanted with 6 different leukemia entities from Icsbp^−/−Nf1^+/− mice. In all cases where LOH was observed in the parental leukemia, this was also observed in the diseased NOD/SCID mice. As a comparison are shown amplicons from genomic DNA from untreated NOD/SCID mice and from normal Nf1^+/+ or Nf1^+/− mice.