MTID: a database of Sleeping Beauty transposon insertions in mice

The Sleeping Beauty (SB) transposon system provides the first random insertional mutagen available for germline genetic screens in mice. In preparation for a large scale project to create, map and manage up to 5000 SB insertions, we have developed the Mouse Transposon Insertion Database (MTID; http://mouse.ccgb.umn.edu/transposon/). Each insertion's genomic position, as well as the distance between the insertion and the nearest annotated gene, are determined by a sequence analysis pipeline. Users can search the database using a specified nucleotide or genetic map position to identify the nearest insertion. Mouse reports describe insertions carried, strain, genotype and dates of birth and death. Insertion reports describes chromosome, nucleotide and genetic map positions, as well as nearest gene data from Ensembl, NCBI and Celera. The flanking sequence used to map the insertion is also provided. Researchers will be able to identify insertions of interest and request mice or frozen sperm that carry the insertion.     

Germline mutagenesis mediated by <Emphasis Type="Italic">Sleeping Beauty</Emphasis>transposon system in mice

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.     

"睡美人"转座子的研究进展

“睡美人( Sleeping Beauty, SB) ”转座系统是Tc1/mariner 转座子超家族中的一员,已经失活了一千多万年。1997年,Ivics 等根据积累的系统发生数据,利用生物信息学的方法, 对其进行分子重建, 终于唤醒了其转座活性。近年来对“睡美人”转座系统的转座效率和转座机理进行的研究,已证明SB转座子在基因筛选,转基因及基因治疗等领域具有广阔的应用前景。文章重点论述了SB转座子在结构及其优化、转座机制和应用等方面的进展,同时对其研究中出现的各种问题进行了总结并提出了一些解决方案。     

Alternative conformations of a nucleic acid four-way junction

Sleeping Beauty (SB), a member of the Tc1/mariner superfamily of transposable elements, is the only active DNA-based transposon system of vertebrate origin that is available for experimental manipulation. We have been using the SB element as a research tool to investigate some of the cis and trans-requirements of element mobilization, and mechanisms that regulate transposition in vertebrate species. In contrast to mariner transposons, which are regulated by overexpression inhibition, the frequency of SB transposition was found to be roughly proportional to the amount of transposase present in cells. Unlike Tc1 and mariner elements, SB contains two binding sites within each of its terminal inverted repeats, and we found that the presence of both of these sites is a strict requirement for mobilization. In addition to the size of the transposon itself, the length as well as sequence of the DNA outside the transposon have significant effects on transposition. As a general rule, the closer the transposon ends are, the more efficient transposition is from a donor molecule. We have found that SB can transform a wide range of vertebrate cells from fish to human. However, the efficiency and precision of transposition varied significantly among cell lines, suggesting potential involvement of host factors in SB transposition. A positive-negative selection assay was devised to enrich populations of cells harboring inserted transposons in their chromosomes. Using this assay, of the order of 10,000 independent transposon insertions can be generated in human cells in a single transfection experiment. Sleeping Beauty can be a powerful alternative to other vectors that are currently used for the production of transgenic animals and for human gene therapy.     

Characterization of Sleeping Beauty transposition and its application to genetic screening in mice

The use of mutant mice plays a pivotal role in determining the function of genes, and the recently reported germ line transposition of the Sleeping Beauty (SB) transposon would provide a novel system to facilitate this approach. In this study, we characterized SB transposition in the mouse germ line and assessed its potential for generating mutant mice. Transposition sites not only were clustered within 3 Mb near the donor site but also were widely distributed outside this cluster, indicating that the SB transposon can be utilized for both region-specific and genome-wide mutagenesis. The complexity of transposition sites in the germ line was high enough for large-scale generation of mutant mice. Based on these initial results, we conducted germ line mutagenesis by using a gene trap scheme, and the use of a green fluorescent protein reporter made it possible to select for mutant mice rapidly and noninvasively. Interestingly, mice with mutations in the same gene, each with a different insertion site, were obtained by local transposition events, demonstrating the feasibility of the SB transposon system for region-specific mutagenesis. Our results indicate that the SB transposon system has unique features that complement other mutagenesis approaches.     

Common physical properties of DNA affecting target site selection of sleeping beauty and other Tc1/mariner transposable elements

Sleeping Beauty (SB) is the most active Tc1/mariner-type transposable element in vertebrates, and is therefore a valuable vector for transposon mutagenesis in vertebrate models and for human gene therapy. We have analyzed factors affecting target site selection of SB in mammalian cells, by generating transposition events from extrachromosomal plasmids to chromosomes. In contrast to the local hopping observed when transposition is induced from a chromosomal context, mapping of 138 unique SB insertions on human chromosomes showed a fairly random genomic distribution, and a 35% occurrence of transposition into genes. Inspection of the DNA flanking the sites of element integration revealed significant differences from random DNA in both primary sequence and physical properties. The consensus sequence of SB target sites was found to be a palindromic AT-repeat, ATATATAT, in which the central TA is the canonical target site. We found however, that target site selection is determined primarily on the level of DNA structure, and not by specific base-pair interactions. Computational analyses revealed that insertion sites tend to have a bendable structure and a palindromic pattern of potential hydrogen-bonding sites in the major groove of the DNA. These features appear conserved in the Tc1/mariner family of transposons and in other, distantly related elements that share a common catalytic domain of the transposase, and integrate fairly randomly. No similar target site preference was found for non-randomly integrating elements. Our results suggest common factors influencing target site selection of a wide range of transposable elements.     

Transposition and gene disruption in the male germline of the mouse

We have tested a synthetic, functional, transposon called Sleeping Beauty for use in mice as a germline insertional mutagen. We describe experiments in which mutagenic, polyadenylation‐site trapping, transposon vectors were introduced into the germline of mice. When doubly transgenic males, expressing the Sleeping Beauty transposase gene (SB10) and harboring poly(A)‐trap transposon vectors, were outcrossed to wild‐type females, offspring were generated with new transposon insertions. The frequency of new transposon insertion is roughly two per male gamete. These new insertions can be passed through the germline to the next generation and can insert into or near genes. We have generated a preliminary library of 24 mice harboring 56 novel insertion sites, including one insertion into a gene represented in the EST database and one in the promoter of the galactokinase (Gck) gene. This technique has promise as a new strategy for forward genetic screens in the mouse or functional genomics. genesis 30:82–88, 2001. © 2001 Wiley‐Liss, Inc.     

Target-site preferences of Sleeping Beauty transposons

The Sleeping Beauty (SB) transposon is a Tc1/mariner family transposon that has applications in vertebrate animals for gene transfer, gene-tagging, and human gene therapy. In this study, we analyzed the target-site preferences of the SB transposon. At the genomic level, integration of SB transposons with respect to genes (exons and introns) and intergenic regions appears fairly random but not on a micro-scale. Although there appears to be a consensus sequence around the vicinity of the target sites, the primary sequence is not the determining factor for target selection. When integrations were examined over a limited topography, the sites used most often for integration did not match the consensus sequence. Rather, a unique deformation inherent in the sequence may be a recognition signal for target selection. The deformation is characterized by an angling of the target site such that the axis around the insertion site is off-center, the rotation of the helix (twisting) is non-uniform and there is an increase in the distance between the central base-pairs. Our observations offer several hypothetical insights into the transposition process. Our observations suggest that particular deformations of the double helix predicted by the V(step) algorithm can distinguish TA sites that vary by about 16-fold in their preferences for accommodating insertions of SB transposons.     

Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells

We describe an experimental approach for generating mutant alleles in rat spermatogonial stem cells (SSCs) using Sleeping Beauty (SB) transposon-mediated insertional mutagenesis. The protocol is based on mobilization of mutagenic gene-trap transposons from transfected plasmid vectors into the genomes of cultured stem cells. Cells with transposon insertions in expressed genes are selected on the basis of activation of an antibiotic-resistance gene encoded by the transposon. These gene-trap clones are transplanted into the testes of recipient males (either as monoclonal or polyclonal libraries); crossing of these founders with wild-type females allows the insertions to be passed to F(1) progeny. This simple, economic and user-friendly methodological pipeline enables screens for functional gene annotation in the rat, with applicability in other vertebrate models where germ line-competent stem cells have been established. The complete protocol from transfection of SSCs to the genotyping of heterozygous F(1) offspring that harbor genomic SB gene-trap insertions takes 5-6 months.     

Retargeting transposon insertions by the adeno-associated virus Rep protein

The Sleeping Beauty (SB), piggyBac (PB) and Tol2 transposons are promising instruments for genome engineering. Integration site profiling of SB, PB and Tol2 in human cells showed that PB and Tol2 insertions were enriched in genes, whereas SB insertions were randomly distributed. We aimed to introduce a bias into the target site selection properties of the transposon systems by taking advantage of the locus-specific integration system of adeno-associated virus (AAV). The AAV Rep protein binds to Rep recognition sequences (RRSs) in the human genome, and mediates viral integration into nearby sites. A series of fusion constructs consisting of the N-terminal DNA-binding domain of Rep and the transposases or the N57 domain of SB were generated. A plasmid-based transposition assay showed that Rep/SB yielded a 15-fold enrichment of transposition at a particular site near a targeted RRS. Genome-wide insertion site analysis indicated that an approach based on interactions between the SB transposase and Rep/N57 enriched transgene insertions at RRSs. We also provide evidence of biased insertion of the PB and Tol2 transposons. This study provides a comparative insight into target site selection properties of transposons, as well as proof-of-principle for targeted chromosomal transposition by composite protein-protein and protein-DNA interactions.     

精子特异性 Sleeping Beauty 转座酶转基因小鼠模型的建立

目的：建立精子特异性表达Sleeping Beauty （ SB）转座酶转基因小鼠模型,为研究SB转座子在小鼠中的应用提供工具。方法克隆精子特异性启动子用以驱动SB转座酶基因的表达,建立精子特异性表达SB转座酶的载体,利用显微注射方法建立以C57BL/6J为背景的精子特异性表达SB转座酶的转基因小鼠。 PCR鉴定首建鼠的基因型,western blot（WB）和免疫组织化学（IHC）检测SB转座酶基因在小鼠生殖腺睾丸中的表达情况,筛选睾丸中高表达SB转座酶的转基因小鼠。结果显微注射方式获得了5只首建小鼠,其中3只能稳定传代,利用WB和IHC成功的筛选出一株在精子中高表达SB转座酶的转基因小鼠。结论成功建立了精子特异性高表达SB转座酶转基因小鼠模型,为将SB转座子作为一种基因工程工具应用于小鼠基因修饰模型的建立提供非常重要的工具资源。     

Sleeping Beauty transposition in the mouse genome is associated with changes in DNA methylation at the site of insertion

The Sleeping Beauty (SB) transposon (Tn) system is a nonviral gene delivery tool that has widespread application for transfer of therapeutic genes into the mammalian genome. To determine its utility as a gene delivery system, it was important to assess the epigenetic modifications associated with SB insertion into the genome and potential inactivation of the transgene. This study investigated the DNA methylation pattern of an SB Tn as well as the flanking genomic region at insertion sites in the mouse genome. The ubiquitous ROSA26 promoter and an initial part of the eGFP coding sequence in the SB Tn exhibited high levels of CpG methylation in transgenic mouse lines, irrespective of the chromosomal loci of the insertion sites. In contrast, no detectable CpG methylation in the endogenous mouse ROSA26 counterpart was observed in the same animals. Furthermore, significant hypomethylation was detected in neighboring chromosomal sequences of two unique SB Tn insertions compared to wild-type patterns. Taken together, these results suggest that SB Tn insertions into the mouse genome can be discriminated by DNA methylation machinery from an identical endogenous DNA sequence and can profoundly alter the DNA methylation status of the transgene cargo as well as flanking host genomic regions.     

Fishing for Answers with Transposons

Transposons are one means that nature has used to introduce new genetic material into chromosomes of organisms from every kingdom. They have been extensively used in prokaryotic and lower eukaryotic systems, but until recently there was no transposon that had significant activity in vertebrates. The Sleeping Beauty (SB) transposon system was developed to direct the integration of precise DNA sequences into chromosomes. The SB system was derived from salmonid sequences that had been inactive for more than 10 million years. SB transposons have been used for two principle uses – as a vector for transgenesis and as a method for introducing various trap vectors into (gene-trap) or in the neighborhood of (enhancer-trap) genes to identify their functions. Results of these studies show that SB-mediated transgenesis is more efficient than that by injection of simple plasmids and that expression of transgenesis is stable and reliable following passage through the germline.     

Excision of Sleeping Beauty transposons: parameters and applications to gene therapy

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase-transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals.     

Structure-function analysis of the inverted terminal repeats of the sleeping beauty transposon

Translocation of Sleeping Beauty (SB) transposon requires specific binding of SB transposase to inverted terminal repeats (ITRs) of about 230 bp at each end of the transposon, which is followed by a cut-and-paste transfer of the transposon into a target DNA sequence. The ITRs contain two imperfect direct repeats (DRs) of about 32 bp. The outer DRs are at the extreme ends of the transposon whereas the inner DRs are located inside the transposon, 165-166 bp from the outer DRs. Here we investigated the roles of the DR elements in transposition. Although there is a core transposase-binding sequence common to all of the DRs, additional adjacent sequences are required for transposition and these sequences vary in the different DRs. As a result, SB transposase binds less tightly to the outer DRs than to the inner DRs. Two DRs are required in each ITR for transposition but they are not interchangeable for efficient transposition. Each DR appears to have a distinctive role in transposition. The spacing and sequence between the DR elements in an ITR affect transposition rates, suggesting a constrained geometry is involved in the interactions of SB transposase molecules in order to achieve precise mobilization. Transposons are flanked by TA dinucleotide base-pairs that are important for excision; elimination of the TA motif on one side of the transposon significantly reduces transposition while loss of TAs on both flanks of the transposon abolishes transposition. These findings have led to the construction of a more advanced transposon that should be useful in gene transfer and insertional mutagenesis in vertebrates.     

The Sleeping Beauty transposable element: evolution, regulation and genetic applications

Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrate species are inactive due to the accumulation of mutations. A representative of a subfamily of fish elements estimated to be last active > 10 million years ago has been reconstructed, and named Sleeping Beauty(SB). This element opened up new avenues for studies on DNA transposition in vertebrates, and for the development of transposon tools for genetic manipulation in important model species and in humans. Multiple transposase binding sites within the terminal inverted repeats, a transpositional enhancer sequence, unequal affinity of the transposase to the binding sites and the activity of the cellular HMGB1 protein all contribute to a highly regulated assembly of SB synaptic complexes, which is likely a requirement for the subsequent catalytic steps. Host proteins involved in double-strand DNA break repair are limiting factors of SB transposition in mammalian cells, underscoring evolutionary, structural and functional links between DNA transposition, retroviral integration and V(D)J recombination. SB catalyzes efficient cut-and-paste transposition in a wide range of vertebrate cells in tissue culture, and in somatic tissues as well as the germline of the mouse and zebrafish in vivo, indicating its usefulness as a vector for transgenesis and insertional mutagenesis.     

Pig transgenesis by Sleeping Beauty DNA transposition

Modelling of human disease in genetically engineered pigs provides unique possibilities in biomedical research and in studies of disease intervention. Establishment of methodologies that allow efficient gene insertion by non-viral gene carriers is an important step towards development of new disease models. In this report, we present transgenic pigs created by Sleeping Beauty DNA transposition in primary porcine fibroblasts in combination with somatic cell nuclear transfer by handmade cloning. Göttingen minipigs expressing green fluorescent protein are produced by transgenesis with DNA transposon vectors carrying the transgene driven by the human ubiquitin C promoter. These animals carry multiple copies (from 8 to 13) of the transgene and show systemic transgene expression. Transgene-expressing pigs carry both transposase-catalyzed insertions and at least one copy of randomly inserted plasmid DNA. Our findings illustrate critical issues related to DNA transposon-directed transgenesis, including coincidental plasmid insertion and relatively low Sleeping Beauty transposition activity in porcine fibroblasts, but also provide a platform for future development of porcine disease models using the Sleeping Beauty gene insertion technology.     

Size matters: versatile use of <Emphasis Type="Italic">PiggyBac</Emphasis> transposons as a genetic manipulation tool

Transposons have been promising elements for gene integration, and the Sleeping Beauty (SB) system has been the major one for many years, although there have been several other transposon systems available, for example, Tol2. However, recently another system known as PiggyBac (PB) has been introduced and developed for fulfilling the same purposes, for example, mutagenesis, transgenesis and gene therapy and in some cases with improved transposition efficiency and advantages over the Sleeping Beauty transposon system, although improved hyperactive transposase has highly increased the transposition efficacy for SB. The PB systems have been used in many different scientific research fields; therefore, the purpose of this review is to describe some of these versatile uses of the PiggyBac system to give readers an overview on the usage of PiggyBac system.     

Gene mutations and genomic rearrangements in the mouse as a result of transposon mobilization from chromosomal concatemers

Previous studies of the Sleeping Beauty (SB) transposon system, as an insertional mutagen in the germline of mice, have used reverse genetic approaches. These studies have led to its proposed use for regional saturation mutagenesis by taking a forward-genetic approach. Thus, we used the SB system to mutate a region of mouse Chromosome 11 in a forward-genetic screen for recessive lethal and viable phenotypes. This work represents the first reported use of an insertional mutagen in a phenotype-driven approach. The phenotype-driven approach was successful in both recovering visible and behavioral mutants, including dominant limb and recessive behavioral phenotypes, and allowing for the rapid identification of candidate gene disruptions. In addition, a high frequency of recessive lethal mutations arose as a result of genomic rearrangements near the site of transposition, resulting from transposon mobilization. The results suggest that the SB system could be used in a forward-genetic approach to recover interesting phenotypes, but that local chromosomal rearrangements should be anticipated in conjunction with single-copy, local transposon insertions in chromosomes. Additionally, these mice may serve as a model for chromosome rearrangements caused by transposable elements during the evolution of vertebrate genomes.     

一种包含转座子Sleeping Beauty和PiggyBac的新型多功能载体的构建

DNA转座子作为一种遗传学工具对脊椎动物的转基因、突变体产生、癌基因发现和基因治疗方面都有巨大的贡献. 目前,哺乳动物中应用最为广泛、活性最高的DNA转座子为重构于鲑鱼的Sleeping Beauty (SB)转座子和来源于甘蓝蠖度尺蛾 (cabbage looper moth Trichoplusia ni)的PiggyBac (PB)转座子. 本研究中,我们成功构建了包含PB和SB两种转座子的杂合转座载体,命名为PBSBD. 在杂合转座载体中融入了基因捕获框及loxp/Frt元件,用以实现转座过程中的基因捕获和条件性敲除. 在HepG2细胞中通过检测报告基因的表达情况及阳性克隆的定位,对构建的杂合转座载体PBSBD进行了活性的初步验证. 结果表明,PBSBD能够有效被2种转座酶识别,并能检测到报告基因的表达. 本研究所构建的杂合转座载体PBSBD结合2种转座酶,可以应用于大规模筛选突变基因和研究基因功能. 并且该杂合转座载体还可以利用SB转座酶的邻近转座特性,结合载体内所包含的loxp/Frt元件用以邻近区域DNA片段的条件性敲除,研究大片段DNA在生物体中的作用.