We have investigated the utility of the SB transposon system as a forward-genetic tool in the mouse germline to recover a range of lethal and viable phenotypes. In a limited number of pedigrees, we recovered one dominant limb and one recessive behavioral phenotype. Unexpectedly, however, genomic rearrangements including deletions and inversions near the transposon donor site, as well as insertion of the donor site plus nearby Chromosome-11 sequences into other chromosomes, caused a high frequency of early embryonic phenotypes in our screen. We hypothesize that the donor site of transposons is situated between these two sets of deletions (B, blue and orange boxes), and that transposition resulted in deletions that were proximal or distal to the donor site. We further hypothesize that sequences contained within these deleted regions are essential, and thus result in the recessive lethal phenotypes we observed. We also observed similar de novo rearrangements in somatic cells. These genetic changes may not have been previously recognized because earlier studies involved sequence- or expression-driven approaches. Through a forward-genetic approach, we identified a high frequency of recessive lethal mutations, and then by complementation testing and molecular analysis, determined the nature of the lesions.
Small- and large-scale local genome rearrangements including insertions, deficiencies, duplications, inversions, and translocations are a common result of Ac/Ds
], Tc1 [23
], and bacterial [26
] element transpositions. Although we have not speculated on the mechanisms by which SB transposons cause rearrangements, the mechanisms for those elements, representing three major families of eukaryotic cut-and-paste transposons, are well-characterized in those reports. Genomic rearrangements caused by alternative transposition of P
] and Ac/Ds
] elements are particularly well understood and the involvement of the latter in altering the structure of Maize
genes is now known [27
]. The SB-induced genomic rearrangements reported here could be caused by alternative mechanisms of transposition, or due to chromosome instability caused by double-strand breakage during excision and integration. In either case, these events may have been exacerbated by the fact that the GT3A donor site consists of many identical copies of the transposon elements and the high mobilization rate in the GT3A strain.
This discovery has implications for using SB and possibly other transposable elements as mutagenic tools in the mouse germline and soma. A recent study took advantage of the local hopping phenomenon of SB transposons to demonstrate regional saturation mutagenesis [5
]. Employing a polyA-trap design, insertions were cloned from sperm and identified in every gene over a 4-Mb interval. The data presented here suggest other genetically linked lesions often accompany insertions in local genes. Indeed, single gene insertions were identified in the cases of the dominant polysyndactyly and recessive hyperactivity, seen in pedigrees BM and BG, respectively. Further analysis, however, led us to discover genomic deletions within the mutagenized Chromosome 11 in each of these pedigrees that are also genetically linked to these phenotypes. Pedigree BM, along with the polysyndactyly phenotype, is a member of complementation group 1 (, B) and harbors a deletion distal to the concatemer donor site. The hyperactive pedigree BG contains two separate deletions (B) in addition to single copy insertions. Since these genomic rearrangements and transposon insertions are genetically linked to the phenotypes we observed, we cannot rule out that the deletions are not causative. As is always the case, it will be crucial to prove that any transposon insertion causes the observed phenotype by remobilizing it from within the gene by re-exposure to transposase, by removing the mutagenic elements of the vector, or transgene rescue. We have previously reported that the germline mobilization rate of single-copy elements is only about 1%, and thus loxP recombination sites were engineered into the T2/GT3/tTA transposon (A), flanking the mutagenic core of the transposon, to potentially rescue expression of a gene [8
Somatic transposition of oncogenic SB transposons is a powerful method for studying the cancer genome of tumors previously inaccessible to retroviral insertional mutagens [6
]. We have aged several GT3A; SB11 mice and have seen no statistically significant increase in mortality compared to controls. It will be important, however, to closely examine whether continuous transposon mobilization leads to genomic rearrangements that can contribute to cancer development in these models. Additionally, although Sleeping Beauty
was the first vertebrate transposon system shown to be active in the germline and somatic cells of mice in vivo, the fly transposon Minos
] and the lepidopteran piggyBac
] were more recently developed for these purposes. Minos,
like SB, belongs to the Tc1/mariner
superfamily of transposons and, although piggyBac
is in a family of its own, due to their cut-and-paste mechanism of mobilization and local hopping activities, they likely have the potential to cause similar genomic rearrangements as we have reported on here. While analyzing the results of mobilizing these vectors, it will be important to include methods to detect these mutations.
Finally, we propose that the genomic rearrangements caused by mobilization of SB transposons may serve as a model for the genomic rearrangements that have occurred during the evolution of vertebrate genomes. Major rearrangements that lead to the observed conserved blocks of synteny between mouse and human chromosomes are consistent with random chromosomal breakage [30
]. Mainstream hypotheses, however, correlate smaller scale rearrangements, including insertion, deletion, inversion, and duplications within chromosomes, with the amplification and activities of transposable elements [31
] though most attention has been given to copy-and-paste elements. To our knowledge, this is the first report of local genomic rearrangements caused by a cut-and-paste transposon in a vertebrate. As far as is known, cut-and-paste transposons have been inactive in mammalian genomes for tens of millions of years [34
]. Nevertheless, cut-and-paste elements do lead to genomic rearrangement in nature, as endemic inversions in isolated populations of Drosophila
have been attributed to hobo
-element cut-and-paste activity [36
]. We have shown here that the high frequency of transposition of the teleost fish-derived SB element [37
] in transgenic mice can cause similar rearrangements at high frequency. Though the synthetic SB element is likely to be much more active than an endogenous cut-and-paste element would have been millions of years ago, we postulate that analogous rearrangements could contribute to the speciation and evolution of vertebrates. Such mutations by a highly active element would undoubtedly be deleterious to a host species over several generations. It now seems plausible that genomes evolved mechanisms to suppress cut-and-paste elements to protect against these damaging rearrangements rather than to prevent the accumulation of rare mutations caused by single-copy insertions.