Large-scale identification of novel potential disease loci in mouse leukemia applying an improved strategy for cloning common virus integration sites.

The identification of common virus integration sites (cVIS) in retrovirally induced tumors in mice provides a powerful strategy to isolate novel transforming genes. Applying virus LTR-specific inverse-PCR and RT-PCR combined with automated sequencing on CasBr-M Murine Leukemia Virus (MuLV) induced myeloid leukemias, 126 virus integration sites were cloned. Using locus- and LTR-specific primers, a nested-PCR/Southern-blotting procedure was developed on genomic DNA from a large panel of MuLV-induced leukemias, to analyse whether a particular virus insertion represented a cVIS. In fact 39 out of 41 integrations analysed this way appeared to represent a common virus integration. We recognized six previously cloned cVISs, i.e. Evi1, Hoxa7, c-Myb, Cb2/Evi11, Evi12, and His1 and 33 novel common insertions, designated Cas-Br Virus Integration Site (Casvis). Among this group we found integrations in or near genes encoding nuclear proteins, e.g. Dnmt-2, Nm23-M2, Ctbp1 or Erg, within receptor genes, e.g. Cb2 or mrc1, novel putative signaling or transporter genes, the ringfinger-protein gene Mid1 and a panel of genes encoding novel proteins with unknown function. The finding that 39 out of 41 integrations analysed represented a cVIS, suggests that the majority of the other virus insertions that were not yet analysed by the PCR/Southern-blotting method are located in a cVIS as well and may therefore also harbor novel disease genes.