Identification of candidate cancer-causing genes in mouse brain tumors by retroviral tagging

Abstract

Murine retroviruses may cause malignant tumors in mice by insertional mutagenesis of host genes. The use of retroviral tagging as a means of identifying cancer-causing genes has, however, almost entirely been restricted to hematopoietic tumors. The aim of this study was to develop a system allowing for the retroviral tagging of candidate genes in malignant brain tumors. Mouse gliomas were induced by a recombinant Moloney murine leukemia virus encoding platelet-derived growth factor (PDGF) B-chain. The underlying idea was that tumors evolve through a combination of PDGF-mediated autocrine growth stimulation and insertional mutagenesis of genes that cooperate with PDGF in gliomagenesis. Common insertion sites (loci that were tagged in more than one tumor) were identified by cloning and sequencing retroviral flanking segments, followed by BLAST searches of mouse genome databases. A number of candidate brain tumor loci (Btls) were identified. Several of these Btls correspond to known tumor-causing genes; these findings strongly support the underlying idea of our experimental approach. Other Btls harbor genes with a hitherto unproven role in transformation or oncogenesis. Our findings indicate that retroviral tagging with a growth factor-encoding virus may be a powerful means of identifying candidate tumorcausing genes in nonhematopoietic tumors.

Retroviral insertional mutagenesis: past, present and future

“…Another approach to fragment separation is to subclone the PCR products directly into a vector, either by cloning gel eluted fragments or by shotgun subcloning the entire mixture (Suzuki et al, 2002;Johansson et al, 2004). Plasmid DNA is isolated from single bacterial colonies and individual clones sequenced, thus allowing complex mixtures of fragments, including those of similar size, to be readily separated.…”

“…Some replication defective strains have also been used to induce tumours by coinfection with a replication competent strain (Jaenisch, 1980;Johansson et al, 2004). The replication competent strain acts as a 'helper' strain, although predictably the competent strain tends to dominate the resulting viremia in these animals, meaning that cloning insertions of the replication defective virus would only yield a subset of the total insertion sites.…”

MicroRNAs and genomic instability

“…For example, mmu-miR-9-1 is found just 4.4 kb proximal of Rhbg; retroviral integration into the 3′ region of the Rhbg gene on mouse chr. 3 is associated with brain tumors in mice [74]. About 52 kb distal to Jak1 on chr.…”

“…6, mmu-miR-29a and mmu-miR-29b-1 reside only 18.6 kb proximal to the Btl6 integration site in an inverted in orientation. Marked by a gene of unknown function, Mo-MuLV integrations on the 3′ side of Bt16 generate lymphomas [77] or brain tumors [74] depending on the mouse model. On the distal end of chr.…”

“…13, interruption of the Fancc gene in the 3′ region or in intron 15 results in brain tumors [74]. A small cluster of miRNAs including the mmu-miR-23b is located 6.4 kb distal to the Fancc gene and therefore, is likely to be disrupted by these retroviral integration events.…”