In contrast to viruses, transposons are not infectious and therefore can propagate only within the host cell. Thus, transposons evolved to cause as less harm as possible. They have low preference for insertion into genes and usually fairly low transpositional activity. Additionally, transposition is effective from conventional plasmids and therefore do not cause any immunological complications [4]. All that make transposons safe, effective tools for genome manipulations that are easy to use and engineer.
However, there are certain basic requirements for any transposon as a genetic tool in any particular model organism. First of all, it is a sufficient level of transpositional activity in a given host. Second, ideally, it has to be neither endogenous copies of that transposon nor it should cross-mobilize any closely related transposons present within a given genome.
Discovery of the Sleeping Beauty
Transposons have been widely used for insertional mutagenesis and germline transgenesis in invertebrate animal models for the past 20 years. The reason for that is a presence of endogenous active transposable elements (TE) and their early discovery: Tc1 element in C. elegans and P element in D. melanogaster [5,6]. However, it was not found any sufficiently active DNA transposons in vertebrate organisms due to the fact that almost all such elements were inactivated millions years ago [7].
First attempt to fill that gap was the idea to make use of invertebrate TE for that purpose. However, it was not that successful. Thus, both nematode element Tc3 and Minos from Drosophila were found to be active in zebrafish and mammalian cells respectively [8,9]. But transposition efficiency remained to be desired.
Another innovative approach in this field was a molecular reconstruction of extinct for over 10 million years transposon from Salmonid fish genome [10]. That element was called the Sleeping Beauty (SB). SB was found to be able to transpose in somatic, embryonic and germinal cells of different vertebrate model organisms [11]. It also didn't show any bias towards integration into genes and was not associated with any signs of proliferative transformation [12]. Thus, SB is much safer than virus-based vectors. SB cargo capacity was estimated up to 10 kb [11] that is sufficient for the most mutagenesis and transgenesis purposes. Furthermore, after identification of a new hyperactive SB transposase (SB100X), SB transposition almost reached efficiency of viral-based methods [13].
Applications
Sleeping Beauty is a highly promising tool for various genome manipulations in vertebrates. SB was found to be useful in loss- and gain-of-function screens, generations of iPSCs and human gene therapy.
Figure 1. Summary of the basic gene trapping strategies. Adopted from Ivics et al. 2009.
Loss-of-function screen. Transposon-based insertional mutagenesis provides a powerful tool for phenotype driven dissection of gene functions in vivo in various vertebrate model organisms. Due to the fact that SB does not show any preference for insertion into genes, various approaches have been utilized to increase its mutagenic effect (Fig. 1). One of the main advantages of transposons over viruses in such screens is that two component binary approach may be utilized. It means that two lines of transgenic animals are used: a "jumpstarter", transposase expressing line and so-called "mutator" line harboring non-autonomous gene trap transposon. Males obtained after their breeding are utilized for crossing with wild-type females. Animals with identified insertions are bred to visualize the recessive phenotype. For in vivo applications mostly fluorescent reporter such as GFP is used. In cell culture systems that approach doesn't not work. Instead transposons are introduced in form of plasmid DNA with a subsequent trans-supplementation of a transposase. In this case drug resistance markers are used as a reporter. Furthermore, in order to obtain bi-allelic mutations special Blm-deficient cell lines are used. They have much higher rates of homology recombination that results in recessive homozygote conversion [14].
Gain-of-function screen. Transposon-based "oncogen trap" cassette (Fig. 1e) may also be used for gene over- or mis-expression. It may lead to development of cancer in experimental animals if either tumor-suppressor or proto-oncogene is targeted. Significant transposon insertions from cancer samples are isolated and novel oncogene candidates may be identified. In contrast to virus-based cancer screen, Sleeping Beauty functions even in non-dividing cells of brain and liver which were not accessible for such screens before.
Generation of iPSCs. Discovery of induced pluripotent stem cells (iPSCs) opened a new era in regeneration medicine. A huge advantage of transposon-based vectors over viral is that reprogramming transgenes can be removed after transformation of cells to a pluripotent state. It can be approached by arresting transposon-transposase complex in "cut" state without subsequent re-insertion.
Human gene therapy. Main features of the Sleeping Beauty such as absence or low immunogenic potential, random genome integration, no rearrangements at the site of integration makes it one of the most suitable vector for gene therapy in human [15]. A transposase can be easily replaced with a gene of interest and resulting construct should be actively delivered into the cells together with a transposase source. For delivery either viral or non-viral techniques can be applied. But non-viral approach looks especially interesting since SB helps to overcome major problems of the non-viral delivery: genome integration and quality of the integrated DNA.
Perspectives
Transposon-based genome manipulation in vertebrates is a fast developing and promising field of a research. Experience adopted from invertebrate model systems gave a rapid raise to transposon-based genome manipulation techniques in vertebrates after discovery of the first sufficiently active SB transposon in these model organisms. However, there are still problems that have to be challenged to improve and optimize SB. First of all, a better understanding of interactions between transposon and cellular environment is required. It is not yet completely clear why transposition efficiencies in some cells are significantly higher than in the cells of other tissue origin [16]. Can SB transpose in any cell type? Can it be influenced or controlled?
Another important issue is a development of more active transposase that are less sensitive for over production inhibition. In future it would an ideal solution to use complexes of preintegrated transposon with transposase. It would solve the problem of over production inhibition and reduce transgene mosaic expression in embryos due to the much earlier integration event.
Even though SB doesn't show any preference for integration into genes, theoretically it can. Therefore, it would be also promising to engineer SB in such a way that it integrates only in safe and desired places within the genome. It is also worth mentioning that other transposon systems active in vertebrates have been developed. Thus, another extinct transposon from frogs was reconstructed named Frog Prince (FP). With growing number of alternative systems their possible applications are also expanding.
Although, there are still many issues to be solved, it is already obvious that transposon-based genome manipulation has become a powerful tool for unravelling the genome mysteries of vertebrate model organisms. There is no doubt that a gap between respective techniques in invertebrates and vertebrates would be bridged soon.
References
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