A modified sleeping beauty transposon system that can be used to model a wide variety of human cancers in mice

Abstract

Recent advances in cancer therapeutics stress the need for a better understanding of the molecular mechanisms driving tumor formation. This can be accomplished by obtaining a more complete description of the genes that contribute to cancer. We previously described an approach using the Sleeping Beauty (SB) transposon system to model hematopoietic malignancies in mice. Here, we describe modifications of the SB system that provide additional flexibility in generating mouse models of cancer. First, we describe a Cre-inducible SBase allele, RosaSBase(LsL), that allows the restriction of transposon mutagenesis to a specific tissue of interest. This allele was used to generate a model of germinal center B-cell lymphoma by activating SBase expression with an Aid-Cre allele. In a second approach, a novel transposon was generated, T2/Onc3, in which the CMV enhancer/chicken beta-actin promoter drives oncogene expression. When combined with ubiquitous SBase expression, the T2/Onc3 transposon produced nearly 200 independent tumors of more than 20 different types in a cohort of 62 mice. Analysis of transposon insertion sites identified novel candidate genes, including Zmiz1 and Rian, involved in squamous cell carcinoma and hepatocellular carcinoma, respectively. These novel alleles provide additional tools for the SB system and provide some insight into how this mutagenesis system can be manipulated to model cancer in mice.

Figure 1. Generation of the RosaSBase^LsL allele

(A) Schematic of ROSA26, RosaSBase, RosaSBase^LsL and RosaSBase^1loxP alleles. A splice acceptor (SA), SB transposase cDNA (SBase) and polyA site (pA) were introduced into the ROSA26 locus to make the RosaSBase allele, as previously described (6). The RosaSBase^LsL allele includes a lox-stop-lox cassette consisting of an EGFP cDNA with three polyA sites flanked by loxP sites (red arrows). This allele is converted to the RosaSB^1loxP allele following Cre recombination. (B) Triple-transgenic mice were aged along with double-transgenic littermates and monitored for tumor formation. Double-transgenic T2/Onc2;RosaSBase^1loxP mice were also aged to determine tumor susceptibility. (C) The types and frequency of tumors are shown for triple-transgenic mice (left) and double-transgenic T2/Onc2;RosaSBase^1loxP mice (right).

Figure 2. Structure and function of the T2/Onc3 transposon

(A) The T2/Onc3 transposon is similar to the earlier T2/Onc2 element except that a CMV_ie enhancer/chicken β-actin promoter (CAG) replaces the MSCV 5’LTR. (B) Survival of T2/Onc3;RosaSBase double-transgenic mice compared to RosaSBase littermates.

Figure 3. Metastasis in T2/Onc3-induced carcinoma

(A) A double-transgenic mouse showed evidence of a hepatocellular carcinoma with multifocal metastasis in the lung (B). (C-D) Histological comparisons were consistent with this finding. Transposon insertion profiles support a shared origin of these tumors as the have two identical insertions in Atp9b and Ube2cbp (Supplementary Table S3).

Figure 4. T2/Onc3 integration into the Rian and Zmiz1 loci

(A) The Rian locus produces a non-coding RNA of unknown function. Embedded within the Rian locus are three miRNA genes (red lines) and a C/D snoRNA cluster in the 3’ end of the gene (not shown). The position and orientation of the major T2/Onc3 insertions are indicated (black arrows). In addition, Donsante et al. identified three AAV integration sites (blue arrows) within the Rian locus that were associated with hepatocellular carcinoma (29). (B) A region containing exons 8–10 of the Zmiz1 locus is shown. All T2/Onc3 insertion events occurred in intron 8 of Zmiz1 in the same orientation as the gene (black arrows). RT-PCR was performed using primers for the T2/Onc3 splice acceptor and donor (blue arrows) with corresponding primers from Zmiz1 exon 8 or 9 (red arrows). (C) Products of the predicted sizes were amplified from 5 tumors with T2/Onc3 insertions at this site. DNA sequencing confirmed the predicted splicing pattern (not shown). However, chimeric Zmiz1 transcripts were not detected in a control tumor that lacks a transposon insertion at this site or in normal skin tissue.