Germline mutagenesis mediated by Sleeping Beautytransposon system in mice
© BioMed Central Ltd 2007
Published: 31 October 2007
Following the descovery of its transposition activity in mammalian culture systems, the Sleeping Beauty (SB) transposon has since been applied to achieve germline mutagenesis in mice. Initially, the transposition efficiency was found to be low in cultured systems, but its activity in germ cells was unexpectedly high. This difference in transposition efficiency was found to be largely dependent on chromosomal status of the host genomic DNA and transposon vector design. The SB transposon system has been found to be suitable for comprehensive mutagenesis in mice. Therefore, it is an effective tool as a forward genetics screen for tagged insertional mutagenesis in mice.
With the completion of the mouse genomic sequencing project, efficient functional genomics is the current and most pressing focus for achieving a better understanding of gene function. Emerging transposon systems have successfully been applied to both germline and somatic cell mutagenesis in mice. These transposon systems have proven useful and should be considered essential tools for functional genomics in mice and other species
Resurrecting the DNA-type Sleeping Beautytransposon and demonstrating its transpositional activity in mammalian cells
In 1997, Ivics and coworkers  reported the important finding that Tc1/mariner type DNA transposase reconstructed from the salmon fish genome had significant transposable activity in mammalian cultured cells. They fittingly named the transposon 'Sleeping Beauty' (SB), because an inactive transposase has been awakened from millions of years of evolutionary sleep. Tc1/mariner superfamily transposases are known to be active in a wide range of species, ranging from protozoa to mammals. However, other members, such as insect (Himar1 and Mos1) and worm (Tc1 and Tc3) transposons, were shown to be much less active in mammalian cultured cells . Initially, the obvious applications of the transposon included its use in germline mutagenesis. However, in the subsequent year Luo and colleagues  reported that chromosomal SB transposition was not efficient in embryonic stem cells, describing an approximate transpositional frequency of about one in 104 cells. If this efficiency is similar to that of germline transposition, then one can easily predict that the efficiency in generating mutant mice would be approximately one mutant out of 104 newborn mice, which is not suitable for applications in high-throughput forward genetics.
Strategy for detecting germline transposition in mice
Efficient transposition in germ cells
According to three published reports on germline SB transposition [2, 4, 8], transpositional frequency of germ cells was approximately 0.1 transposition/transposon per germ cell, which was about 1,000-fold higher than that of cultured cells. If the original donor site contains ten transposon copies, then each germ cell bears approximately one transposition site. Therefore, the frequency of transposition in germ cells is suitable for comprehensive mutagenesis in mice.
The other important issue would be how many different mutant mice can be generated from a double transgenic mouse. To determine the complexity, we directly analyzed spermatozoon DNA from the double transgenic mice. Because individual transposon integration sites were detected at the frequency of one in 10,000 spermatozoon and each spermatozoon contained approximately one transposed DNA, the complexity of transposition in germ cells was approximately 10,000 . This means that 10,000 different mutant mice could be produced from a single double transgenic mouse, which represents a sufficiently large number of transposition events and renders this technology suitable for use in large scale forward genetics research.
Heterochromatin status affects transposition efficiency
That higher transposition frequencies were observed in mice compared with culture systems may be explained by the epigenetic status of transposons. To investigate this issue further, methylated or unmethylated transposon DNA was introduced into cultured cells . The methylation status of both transposon vectors used was confirmed after integration into the genome; SB transposase was subsequently introduced and rates of excision frequency were determined . Excision frequency was enhanced approximately 100-fold using methylated transposon DNA compared with unmethylated DNA. Methylated transposon DNA also introduced histone modifications such as histone H3-K9 tri-methylation, which is a marker of heterochromatin status .
Recently, the influence of heterochromatin conformation on SB transposition was further examined by recruiting a tetracycline-controlled transrepressor (tTR), which induces heterochromatin conformation, to the SB transposon through a tet operator sequence . Chromatin immuno-precipitation analysis revealed that SB transposase was clearly bound to the tTR induced heterochromatin conformation. The frequency of SB transposition was simultaneously enhanced by approximately 100-fold. These findings indicate that the high affinity of SB transposase for heterochromatin conformation results in enhanced transposition efficiency.
Construction of Sleeping Beautytransposon vector for germline mutagenesis
We found that more than half of transposon integration sites were mapped to the same chromosome bearing the original donor site (transposon concatemer), and preferential transposition occurring near to the original donor site has been clearly demonstrated . Remaining transposition events were widely and randomly distributed throughout the mouse genome, with no apparent preferential integration sites . Therefore, SB transposon system can be utilized for both region specific and genome wide germline mutagenesis.
Generation of mutant mice using the Sleeping Beautytransposon system
Using a new version of the transposon vector, GFP is activated only if the transposed transposon vector is reintegrated into endogenous genes in a sense orientation. Two different transgenic mice bearing this transposon vector were generated; one has a 20-copy transposon concatemer on chromosome 12 and the other has approximately 100 copies on chromosome 7. Approximately 7% of newborn mice from both double transgenic mice derived from these two different transposon bearing mice were GFP positive, suggesting that copy number and chromosomal location of the transposon are not the major determinants of transposition efficiency. This noninvasive GFP examination allowed us to focus on potential mutants soon after birth. Mutant mice obtained from two different donor sites were extensively analyzed. It was found that region specific saturation mutagenesis was possible within a 4 megabase region of the original donor site, and remaining transpositions were widely and randomly distributed, as indicated by the first version of the transposon vector . We compiled a database of SB transposon insertion sites identified in GFP positive progeny and germ cells from double positive transgenic mice . This database is available online  and sperm from many of these lines were stocked in liquid nitrogen.
In the second strategy, by focusing on transpositions with chromosomes that bear no donor site, we can easily avoid the donor site effect (Figure 7, right). In certain transgenic mice with a mobilized transposon vector, more than half of such transpositions occurred on chromosomes other than the original donor site . After segregating the donor site from these mice, we will be able to generate mutant mice in a comprehensive manner without the donor site effect.
The SB transposon system is an effective tool as a forward genetics screen for tagged insertional mutagenesis in mice. There are many advantages of the SB transposon system relative to other mutagenesis approaches, which include the following: no embryonic stem cell manipulation is required; a simple breeding scheme allows generation of mutant mice; noninvasive screening and rapid identification of disrupted genes are possible by using the transposon sequences as a DNA tag; and, finally, region specific saturation mutagenesis is possible.
Currently, we and others have shown that this system works as an effective insertional mutagenesis screen in both germline and somatic cells [17, 19–21]. An improvement to the SB transposon system for germline mutagenesis would be to increase the number of gene hits, in order to make the system more attractive to the forward genetics community and better suited to comprehensive mutagenesis screening. This can be done in many ways, including the following: optimize the transposon cargo cassette as transposition efficiency decreases with increasing vector size; screen for more effective hyperactive mutants of SB transposase for increasing transposition events; and, finally, introduce transposon vectors that can alter heterochromatin conformation change within the donor site to increase transposition efficiency.
Because SB transpositions may cause deleterious effects at the donor site concatemers , careful phenotypic analysis of mutant mice progeny is required, taking into consideration the possibility of a donor site effect. To overcome this limitation of SB transposition, donor sites containing single copies of SB transposon may be used. Better versions of the SB transposon system with high efficiency of transposition would make this a viable option .
Since the development of the SB transposon system, several other transposon systems have been reported to be active in mammalian cells, including Minos  (isolated from Drosophila hydei), piggyBac  (from the moth Trichoplusia), and Tol2  (from the Japanese medaka fish Oryzias latipes). Recently, Balciunas and coworkers  reported that Tol2 transposase can efficiently transpose DNA sequence larger than 10 kilobases, suggesting its possible use for gene therapeutic and transgenic applications. Although further studies are needed to clarify the characteristics of these transposons, the diversity of the available tools will greatly facilitate future applications of tagged mutagenesis to functional analysis of the genome.
We thank C Kokubu, K Yusa, M Kouno, K Yae, R Ikeda, ES Saito, Y Uno, T Hayakawa, S Kouno, and Y Odan for their advice and excellent support. This work was supported by grants from the New Energy and Industrial Technology Development Organization of Japan; RIKEN, The Institute of Physical and Chemical Research; and a grant-in-aid for Science Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
This article has been published as part of Genome Biology Volume 8, Supplement 1, 2007: Transposons in vertebrate functional genomics. The full contents of the supplement are available online at http://genomebiology.com/supplements/8/S1.
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