Insertional mutagenesis of preneoplastic astrocytes by Moloney murine leukemia virus

Retroviral infection can induce transcriptional activation of genes flankin g the sites of proviral integration in target cells. Because integration is essentially random, this phenomenon can be exploited for random mutagenesi s of the genome, and analysis of integration sites in tumors may identify p otential oncogenes. Here we have investigated this strategy in the context of astrocytoma progression. Neuroectodermal explants from astrocytoma-prone GFAP-v-src transgenic mice were infected with the ecotropic Moloney murine leukemia virus (Mo-MuLV). In situ hybridization and FAGS analysis indicate d that astrocytes from E12.5-13.5 embryos were highly susceptible to retrov iral infection and expressed viral RNA and proteins both in vitro and in vi vo. In average 80% of neuroectodermal cells were infected in vitro with 9-1 4 proviral integrations per cell. Virus mobility assays confirmed that Mo-M uLV remained transcriptionally active and replicating in neuroectodermal pr imary cultures even after 45 days of cultivation. Proviral insertion sites were investigated by inverse long-range PCR. Analysis of a limited number o f provirus flanking sequences in clones originated from in vitro infected G FAP-v-src neuroectodermal cells identified loci of possible relevance to tu morigenesis. Therefore, the approach described here might be suitable for a cceleration of tumorigenesis in preneoplastic astrocytes. We expect this me thod to be useful for identifying genes involved in astrocytoma development /progression in animal models.