The Tc1/mariner family is a large family of widely distributed "cut-and-paste" transposable elements flanked by inverted terminal repeats, which in some cases themselves have embedded direct repeat sequences (for review see [
1]). These elements have been found in all vertebrate genomes examined, but are in all cases defective, containing frameshift mutations, stop codons and small or large deletions in the transposase gene. A lot of research has been invested into this family of transposons based upon investigations into the active proto-typical Tc1, from
Ceanorhabditis elegans, and mariner elements, such as Mos1 from
Drosophila mauritania. Members of this family contain a transposase with a DNA binding domain distantly related to the paired-box DNA binding domain. In addition, a catalytic DDE domain has been identified and critical amino acids in this domain have been discovered [
1].
The mariner and Tc1 transposases have been purified, and remarkably, can be combined with a transposon DNA substrate, which will then undergo transposition
in vitro without a requirement for any other proteins [
35,
36]. Two closely related versions of mariner have been well studied, Mos1 and Himar1, but are widespread in insects [
37]. The Himar1 element is a consensus sequence based on several clones isolated from the horn fly,
Haematobia irritans, and Mos1 was isolated from
Drosophila mauritania [
35]. The wide distribution of Tc1/mariner transposons, the ability of mariner to function in vitro in purified form and in distantly related species, suggested that such elements might require no co-factors for transposition in cells, or use very highly conserved host factors. For example, Mos1 has been introduced into the distantly related insect species, such as the yellow fever mosquito
Aedes aegypti [
38]. For these reasons, great enthusiasm exists for adapting Tc1/mariner-like transposons for use in vertebrate species. Indeed mariner, Tc1 and Tc3 show activity in cultured mouse and human cell lines [
39,
40]. A screen for hyperactive mutants in
E. coli resulted in the identification of amino acid substitutions that improve the activity of Himar1 in cultured cells [
41]. The Mos1 mariner transposon has been used to achieve germline transgenesis in the chicken [
5]. Both mariner and Tc3 has been used to achieve germline transgenesis in zebrafish [
42,
43]. The purified Himar1 transposase efficiently catalyzes interplasmid transposition [
35]. The same kind of interplasmid transposition assay was used in a human 293T embryonic kidney cell line, showing that Himar1 transposition can occur in mammalian cells [
44]. While Himar1 is primarily used for insertional mutagenesis in bacteria and mycobacteria, its potential for vertebrate gene transfer and insertional mutagenesis remains unclear. It remains to be determined if the active mariner elements, Tc1, or Tc3 can be used to mobilize chromosomally resident transposon vectors in vertebrates.
The Minos element, a Tc1/mariner-like transposon from
Drosophila hydei, has been shown to be active in cultured human cell lines [
45,
46]. A gene-trap Minos transposon has been constructed and a library of insertions in HeLa cells were obtained [
46]. The Minos transpoase gene has been expressed in transgenic mice from B cell lineage specific and male sperm cell specific promoters [
47,
48]. When these mice were crossed with transgenic mice carrying Minos transposon vectors, transposition occurred in B cells and in the male germline respectively. Germline insertions occurred in rough one in every ten offspring. Several germline insertions were cloned and each was found to be on a different chromosome [
48]. These data suggest that Minos might be useful for mouse germline mutagenesis and that no distinct preference for local transposition occurs using this transposon system. Thus, genome-wide insertional mutagenesis with Minos is a real possibility in the mouse and possibly other species as well.
The
Sleeping Beauty transposon (SB) is a synthetic Tc1/mariner family transposon derived from defective elements cloned from various Salmonid fish genomes [
30]. The development of SB is particularly significant because it is the first vertebrate transposable element reconstructed from defective endogenous elements. This was accomplished by the stepwise repair of the open reading frame, nuclear localization signal, DNA binding domain, and catalytic activity. Because this process required ten major steps, the SB transposase was designated SB10. SB has been extensively studied. A model of the SB transposition reaction has been proposed based on studies of its inverted terminal repeat structures [
49]. As part of this study an improved inverted terminal repeat sequence, designated pT2, was discovered [
49]. SB related transposons have complicated inverted terminal repeats, each with two embedded direct repeats called the inner direct repeats and the outer direct repeats. The direct repeats, which are ~25 base pairs long, are the sites of transposase binding [
30]. Neither the inverted repeats nor the direct repeats are perfect, and clear differences exist between the right and left inverted repeats and between the inner and outer direct repeats. The rules governing the transposition are not completely understood, but higher binding activity does not translate into increased transposition [
49]. In addition, continued examination of the SB10 transposase sequence led to the identification of additional amino acid substitutions that confer increased activity in gene transfer into transfected HeLa cells [
50]. SB transposons have been used to achieve germline transgenesis in the mouse [
4]. In these experiments, one-cell mouse embryo pronuclei were co-injected with transposon vector DNA and in vitro transcribed mRNA encoding the SB10 transposase. The overall transgenesis rate was increased 1.5 fold over background without transposase. The increase in transgenesis rate was entirely due to offspring with multiple, independent transposon insertions. An average of three insertions per animal was obtained in some experiments and transposon transgenes could be expressed. However, the transgenes within transposon vectors seem to be subject to position effect variagation, just as are standard mouse transgenes. These data showed that mammalian germline transgenesis by transposition is possible and suggest that the SB system might find utility in other mammalian species in which transgenesis is difficult. Another application of SB has been somatic cell transgenesis. SB can be used for stable, long-term gene transfer and expression into the liver of adult mice [
51]. In some experiments, the SB transposon vector and transposase transgene have been delivered by so-called "hydrodynamic therapy". In this approach, 10% of the weight of the mouse of DNA-containing Ringer's solution is injected via the tail vein into mice in less than ten seconds. For the average mouse this is roughly 2–2.5 milliliters. The resultant transient increase in venous pressure within the liver is thought to result in extravasation of the plasmid DNA, which is efficiently taken up by ~25% of hepatocytes [
52]. The Factor IX gene was cloned into an SB transposon vector and delivered to hemophilia B knockout mice using this technique by Dr. Mark Kay's laboratory [
51]. Long-term expression required co-delivery of the Factor IX transposon and an active SB10 transposase gene on another plasmid. Similar success has been achieved for long-term gene transfer of the FAH gene into knockout mouse liver [
53], and the
LAMB3 gene into cultured human skin cells from patients with junctional epidermolysis bullosa syndrome to correct this disorder in xenografted nude mice [
54]. Dr. Mark Kay's group also has developed "binary" gene therapy vectors in which both the SB transposon vector and SB10 transposase gene were delivered to hepatocytes using adenoviral vectors, which by themselves only result in transient gene transfer and expression [
31]. Interestingly, efficient gene transposition required recombinase-mediated excision and circularization of the transposon vector from the linear adenoviral DNA, suggesting that circularized transposon vector DNA is more efficiently transposed, at least in this environment [
31]. Dr. Scott McIvor's laboratory has developed methods for long term gene transfer and expression into adult mouse lung using SB vector and SB10 transposase DNA complexed with polyethyleneimine (PEI), a polycationic, branched molecule (Beleur et al., Molecular Therapy, In Press). Finally, in the area of germline insertional mutagenesis, we have demonstrated that SB transposons present in chromosomes of SB transposase transgenic mice will efficiently undergo transposition in germline cells, such that offspring from these mice are obtained with new transposon insertions [
55]. Similar results were published in 2001 by two other labs using SB, one in Japan and one in the Netherlands [
19,
56]. Three papers all report essentially similar results, with differences in the average number of new transposon insertions obtained. The ubiquitously expressed CAGGS [
55,
56], or the male germline specific protamine 1 promoters [
19] were used to drive expression of the SB transposase. Single copy [
19] or multicopy transposon vectors were used, all with different internal sequences in the transposon itself. An average of 0.2 [
19], 1 [
56], or 2.0 [
55] new transposon insertions per offspring were obtained. These data clearly establish that the SB system can be used to achieve high efficiency transposition in the male or female germline. When we bred animals with new transposon insertions they were present in roughly half of the offspring of these animals and could segregate independently as if they had transposed to multiple chromosomes. Moreover, if the animal bred was also transgenic for the SB10 transposase, then a number of new transposon insertions were detected. It is important to note that offspring are generated with as many as 11 new transposon insertions, when the transposon concatomer is passed through the germline twice in the presence of the transposase transgene (unpublished data). To be useful as insertional mutagens, SB transposon vectors should be capable of inserting into genes. Indeed, transposon vector insertion into genes has been observed in primary mouse liver cells [
51] and in cultured cell lines. Using inverse PCR or a linker-mediated PCR technique [
55] we have cloned and sequenced 44 germline transposon insertions and analyzed them and mice carrying these insertions (Carlson et al., Genetics, In Press). All the insertions cloned are flanked by TA dinucleotides as expected for Tc1/mariner family transposition. Analysis of the transposon insertions showed that the adjacent plasmid sequence from the concatomer had been replaced by mouse genomic sequence as expected if true transposition had occurred. The distribution and sequence content flanking these cloned insertion sites was compared to 44 mock insertion sites randomly selected from the genome. We found that germline SB transposon sites are AT-rich and the sequence ANNTANNT is favored compared to other TA dinucleotides. Local transposition occurs with insertions linked to the donor site roughly 40% of the time. The size of this local hopping interval is roughly 3–5 cM or 10–12 Mbp. We find roughly 30% of the transposon insertions are in transcription units as determined using the Celera database, similar to the percentage of random TA dinucleotides. We also determined that transposons inserted within a gene, in the same orientation as the gene, are subject to splicing from upstream exons of the endogenous gene. Significantly, we now know how often transposon insertions occur within genes (~25–30% of the time), how often a transposon insertion occurs locally near the donor site (~50% of the time), and how big the local region is for SB-mediated transposition (~3–5 cM or 10–15 Mbp). The results are all consistent with the use of SB for forward genetic screens in local intervals of the mouse genome. Thus, assuming 2 new transposon insertions per gamete (as we have already achieved) we can expect to achieve a 1X coverage of a 10 Mbp region of the genome (with one insertion every 10 kb) in as few as 1000 mice. We have begun to characterize two embryonic lethal mutations caused by endogenous splicing disruption in mice carrying intron-inserted SB gene-trap transposons. It is clear from these analyses that SB and probably other cut-and-paste transposons have potential utility as random germline mutagens for forward genetic screens.