In this report we describe the generation of a Cre-inducible Sleeping Beauty
transposon system that eliminates the embryonic lethality associated with the original SB system (6
), and allows tissue-specific control of SB transposase expression, modifying the tumor spectra produced. In the absence of a Cre transgene, the SB transposase is not expressed and tumors are not generated. This allows us to maintain mouse strains that are double homozygous for the conditional RosaSBaseLsL
allele and a mutagenic transposon donor transgene. These mice can be used to generate large cohorts of tumor-prone animals through a single cross to a tissue-specific Cre transgenic line, and have already been employed to generate models of colorectal cancer and hepatocellular carcinoma (11
). Here, we use an Aid-Cre allele to activate transposon mutagenesis in germinal center B-cells.
Finally, we show that altering the structure of the mutagenic SB transposon can have a profound effect on the tumor types induced by transposition. While both are ubiquitous promoters, MSCV is very efficient in hematopoietic cells and CAG works efficiently in epithelial cells (24
). By replacing the MSCV LTR in the original T2/Onc2 transposon with the CAG promoter, we shifted the tumor spectrum from mostly lymphomas to primarily carcinomas. This suggests the promoter used to generate gain-of-function mutations in oncogenes upon SB integration significantly impacts the tumor types produced. This further implies that the tumor spectrum generated by SB mutagenesis could potentially be altered by placing tissue-specific promoters within the mutagenic transposon. When combined with the Cre-inducible RosaSBaseLsL
allele, transposon promoter variants could provide even finer control of Sleeping Beauty
mutagenesis in vivo
The SB-induced carcinoma model described is highly significant, not only because of its ability to generate solid tumors, but also because of its ability to produce metastatic tumors. While the metastasis rate was low, it should be noted that each animal had an average of 3 independent tumors at the time of sacrifice, and high tumor burden may have caused the animals to become moribund before any macroscopic metastatic lesions could develop. Restricting SB mutagenesis to a specific tissue using the conditional RosaSBaseLsL
allele could potentially increase the rate of metastasis. An alternative approach would be to induce SB mutagenesis in a transgenic or knockout strain background that develops spontaneous tumors with low metastatic potential, in order to identify mutations that accelerate metastasis. The SB system could then be used to study metastasis by identifying genes that contribute to a metastatic cell’s ability to survive and proliferate outside of the primary tumor site (25
The power of forward genetic screens using SB transposition is greatly enhanced by larger tumor panels. Unfortunately, the types of carcinoma that developed in mice with the transposon carrying the CAG promoter were so diverse that we were unable to obtain significant numbers of tumors for most tumor types. However, we were able to identify novel candidate cancer genes for squamous cell carcinoma of the skin and hepatocellular carcinoma, the two most common tumor types in these mice. The limited number of samples is likely the reason we did not identify more candidate cancer genes in these two tumor types. The genomic landscapes of SB-induced tumors and human tumors are similar in that relatively few candidate cancer genes are mutated by SB transposon insertion in a large percentage of tumors. These genes can be easily identified in even small tumor panels (i.e. Zmiz1, Rian). Analysis of large SB-induced tumor panels will be required to identify candidate cancer genes that appear as gene hills on the cancer genomic landscape.
The SB system we describe here demonstrates the flexibility and power of this system for identifying novel cancer genes in many tumor types. Our work also suggests that SB transposition is able to model many forms of human cancer in mice, including carcinomas. Thus, the SB system offers a complementary approach in the identification of cancer genes and may play an important role in deciphering the genetic complexity of human tumors. The SB system will likely identify a set of candidate genes that partially overlaps those identified in human tumors. Genes identified by both approaches are much more likely to represent cancer genes and would merit further study. Additionally, the SB transposition system could be used to address basic biological questions, such as metastasis or resistance to therapeutic agents, that would be difficult to address directly in human cancer patients.