Transposon-mediated mutagenesis of somatic cells in the mouse for cancer gene identification

Abstract

To successfully treat cancer we will likely need a much more detailed understanding of the genes and pathways meaningfully altered in individual cancer cases. One method for achieving this goal is to derive cancers in model organisms using unbiased forward genetic screens that allow cancer gene candidate discovery. We have developed a method using a "cut-and-paste" DNA transposon system called Sleeping Beauty (SB) to perform forward genetic screens for cancer genes in mice. Although the approach is conceptually similar to the use of replication competent retroviruses for cancer gene identification, the SB system promises to allow such screens in tissues previously not amenable to forward genetic screens such as the gastrointestinal tract, brain, and liver. This article describes the strains useful for SB-based screens for cancer genes in mice and how they are deployed in an experiment.

Fig. 1

The T2/Onc transposon vector. This vector contains elements designed to elicit either transcriptional activation (MSCV 5′ LTR and splice donor [SD]) or inactivation (splice acceptors [SA] and polyadenylation signals [pA]). The position of primers used to detect T2/Onc in transgenic mice is shown.

Fig. 2

Typical crosses to generate tissue-specific mobilization of T2/Onc in mice. (A) Tissue-specific Cre transgenic mice (TSP-Cre) are crossed to mice doubly homozygous for a T2/Onc multi-copy transgene (T2/Onc) and the Cre/LoxP—regulated, or conditional, Rosa26 SB11 “knock-in” allele designated RosaSB^LSL. The desired experimental class offspring are triply transgenic. (B) The same cross could also be performed using a parent transgenic for a TSP-Cre transgene and a cancer predisposing mutation or transgene, here referred to generically as Tg.

Fig. 3

Photomicrographs of immunohistochemical detection of SB transposase protein. Images from formalin fixed paraffin embedded sections from the cerebrum and cerebellum are shown in the top and bottom images respectively. Sections were stained with anti-SB transposase antibody (brown stain) and counterstained with Phoenix blue nuclear stain. Mice carrying Cnp-Cre; RosaSB^LSL; T2/Onc (the chromosome 15 concatemer from [5]) transgenes were the source of the tissues in the two right-hand images and control mice which do not carry a Cre transgene, but carry the other two transgenes, are shown in the two left-hand images. Strong nuclear staining is observed in a subset of cells in both tissues in triple transgenic mice, but not Cre-negative control tissues.