Genetic profile of insertion mutations in mouse leukemias and lymphomas

Abstract

Murine leukemia retroviruses (MuLVs) cause leukemia and lymphoma in susceptible strains of mice as a result of insertional mutation of cellular proto-oncogenes or tumor suppressor genes. Using a novel approach to amplify and sequence viral insertion sites, we have sequenced >200 viral insertion sites from which we identify >35 genes altered by viral insertion in four AKXD mouse strains. The class of genes most frequently altered are transcription factors, however, insertions are found near genes involved in signal transduction, cell cycle control, DNA repair, cell division, hematopoietic differentiation, and near many ESTs and novel loci. Many of these mutations identify genes that have not been implicated in cancer. By isolating nearly all the somatic viral insertion mutations contributing to disease in these strains we show that each AKXD strain displays a unique mutation profile, suggesting strain-specific susceptibility to mutations in particular genetic pathways.

Figure 1

VISA PCR using MuLV LTR-specific primers. (a) Schematic representation of VISA. Viral insertion sites are amplified using primers specific for the long terminal repeat (LTR) of the MuLV in combination with RE-anchored degenerate primers that amplify from flanking genomic DNA. Two rounds of PCR are performed on unmodified genomic DNA using nested virus-specific primers to ensure specificity. Successful amplification of any viral insertion site is dependent on whether the RE anchor sequence is present in the genomic sequence flanking the viral LTR. Therefore, to increase the number of insertion mutations amplified, a minimum of five RE-anchored degenerate primers are used on each sample. The approximate binding sites for LTR specific primers SP1 and SP2 are shown. The RE-anchored degenerate primer contains an M13F linker (shown as a curved line) that is not expected to bind mouse DNA during the first round of PCR, but is useful for subsequent amplifications and sequencing. The stripped box indicates the location of the restriction enzyme recognition site target for the RE-anchored degenerate primer. (b–d) Representative examples of VISA products generated with RE-anchored primers specific for HindIII (b), EcoRI (c), and BclI (d). DNA isolated from brain tissue, which is rarely infiltrated by tumor cells, was used as a control to identify all nontarget amplification products such as endogenous proviral insertion sites, internal provirus sequences, or single primer amplification products generated by the restriction-site anchored primer. Tumor-specific VISA products, ranging in size from 50 bp to 2.0 Kb were gel purified and sequenced directly (identified by arrows). Lane designations are (n) (nontumor); (t) tumor.

Figure 2

Meiotic mapping of Lvis2–Lvis4. Lvis viral insertion sites were mapped using an (SB/Le × M. spretus) F₁ × SB/Le interspecific backcross previously typed for multiple markers on all mouse chromosomes (Justice et al. 1990). Partial linkage maps of chromosomes 7 (a), 13 (b), and 12 (c) are depicted. Ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci (with map distances in centimorgans ± the standard error) are shown to the left of each chromosome. For markers that cosegregate, the upper 95% confidence interval is shown in parentheses. The predicted map locations of human orthologs are indicated to the right of each chromosome. References and additional information regarding human map locations can be obtained from the Mouse Genome Database (http://www.informatics.jax.org/).