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.     

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.     

Enhancement of Sleeping Beauty transposition by CpG methylation: possible role of heterochromatin formation

The Sleeping Beauty (SB) transposase is the most active transposase in vertebrate cells, and the SB transposon system has been used as a tool for insertional mutagenesis and gene delivery. Previous studies have indicated that the frequency of chromosomal transposition is considerably higher in mouse germ cells than in mouse embryonic stem cells, suggesting the existence of unknown mechanisms that regulate SB transposition. Here, we demonstrated that CpG methylation of the transposon region enhances SB transposition. The transposition efficiencies of a methylated transposon and an unmethylated transposon which had been targeted in the same genomic loci by recombination-mediated cassette exchange in mouse erythroleukemia cells were compared, and at least a 100-fold increase was observed in the methylated transposon. CpG methylation also enhanced transposition from plasmids into the genome. Chromatin immunoprecipitation assays revealed that histone H3 methylated at lysine-9, a hallmark of condensed heterochromatin, was enriched at the methylated transposon, whereas the unmethylated transposon formed a relaxed euchromatin structure, as evidenced by enrichment of acetylated histone H3 and reporter gene expression. Possible roles of heterochromatin formation in the transposition reaction are discussed. Our findings indicate a novel relationship between CpG methylation and transposon mobilization.     

Regional insertional mutagenesis of specific genes on the CIC5F11/CIC2B9 locus of Arabidopsis thaliana chromosome 5 using the Ac/Ds transposon in combination with the cDNA scanning method.

We have recently developed a novel cDNA selection method (the cDNA scanning method) to select cDNAs for expressed genes in specific regions of the genome [Hayashida et al. (1995) Gene 165: 155, Seki et al. (1997) Plant J. 12: 481]. The gene Ds is known to transpose mainly in its neighborhood. By combining the cDNA scanning method with this trait of Ds, we started functional analysis of region-specific expressed genes on the Arabidopsis thaliana genome. DNA fragments of yeast artificial chromosome (YAC) clones CIC5F11 and CIC2B9 on A. thaliana chromosome 5 were used for the selection of region-specific cDNAs. In total, 50 and 68 cDNA clones were selected from CIC5F11 and CIC2B9, respectively. In parallel, we transposed Ds from a donor T-DNA line, which was mapped on the CIC5F11/CIC2B9 locus of chromosome 5, and obtained Ds-transposed lines. To isolate Ds insertion mutants in the 10 specific genes identified by the cDNA scanning method, we carried out PCR-based screening of 100 Ds-transposed lines and found that 2 lines contain Ds mutations in the genes isolated. We also isolated Ds-flanking genomic DNAs by thermal asymmetric interlaced PCR (TAIL-PCR) in 153 Ds transposon-tagged lines. Southern blot analysis showed that 14% of the lines contained the transposed Ds in the CIC5F11/2B9 region. This suggests that this Ac/Ds transposon system is effective for region-specific insertional mutagenesis.     

Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon

Background  

Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB) transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern.     

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.     

Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region.

Rhodobacter capsulatus mutants unable to fix nitrogen were isolated by random transposon Tn5 mutagenesis. The Tn5 insertion sites of 30 Nif- mutants were mapped within three unlinked chromosomal regions designated A, B, and C. The majority of Tn5 insertions (21 mutants) map within nif region A, characterized by two ClaI fragments of 2.5 and 25 kilobases (kb). The 17-kb ClaI fragment of nif region B contains six nif::Tn5 insertions, and the three remaining mutations are located on a 32-kb ClaI fragment of nif region C. Hybridization experiments using all 17 Klebsiella pneumoniae nif genes individually as probes revealed homology to nifE, nifS, nifA, and nifB in nif region A. The nifHDK genes were localized in nif region B. About 2 kb away from this operon, a second copy of the DNA fragments homologous to nifA and nifB, originally found in nif region A, was identified.     

Genome-wide insertional mutagenesis in human cells by the Drosophila mobile element Minos

The development of efficient non-viral methodologies for genome-wide insertional mutagenesis and gene tagging in mammalian cells is highly desirable for functional genomic analysis. Here we describe transposon mediated mutagenesis (TRAMM), using naked DNA vectors based on the Drosophila hydei transposable element Minos. By simple transfections of plasmid Minos vectors in HeLa cells, we have achieved high frequency generation of cell lines, each containing one or more stable chromosomal integrations. The Minos-derived vectors insert in different locations in the mammalian genome. Genome-wide mutagenesis in HeLa cells was demonstrated by using a Minos transposon containing a lacZ–neo gene-trap fusion to generate a HeLa cell library of at least 10⁵ transposon insertions in active genes. Multiple gene traps for six out of 12 active genes were detected in this library. Possible applications of Minos-based TRAMM in functional genomics are discussed.     

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.     

Generation of an inducible and optimized piggyBac transposon system

Genomic studies in the mouse have been slowed by the lack of transposon-mediated mutagenesis. However, since the resurrection of Sleeping Beauty (SB), the possibility of performing forward genetics in mice has been reinforced. Recently, piggyBac (PB), a functional transposon from insects, was also described to work in mammals. As the activity of PB is higher than that of SB11 and SB12, two hyperactive SB transposases, we have characterized and improved the PB system in mouse ES cells. We have generated a mouse codon-optimized version of the PB transposase coding sequence (CDS) which provides transposition levels greater than the original. We have also found that the promoter sequence predicted in the 5′-terminal repeat of the PB transposon is active in the mammalian context. Finally, we have engineered inducible versions of the optimized piggyBac transposase fused with ERT2. One of them, when induced, provides higher levels of transposition than the native piggyBac CDS, whereas in the absence of induction its activity is indistinguishable from background. We expect that these tools, adaptable to perform mouse-germline mutagenesis, will facilitate the identification of genes involved in pathological and physiological processes, such as cancer or ES cell differentiation.     

Sleeping beauty transposase has an affinity for heterochromatin conformation

The Sleeping Beauty (SB) transposase reconstructed from salmonid fish has high transposition activity in mammals and has been a useful tool for insertional mutagenesis and gene delivery. However, the transposition efficiency has varied significantly among studies. Our previous study demonstrated that the introduction of methylation into the SB transposon enhanced transposition, suggesting that transposition efficiency is influenced by the epigenetic status of the transposon region. Here, we examined the influence of the chromatin status on SB transposition in mouse embryonic stem cells. Heterochromatin conformation was introduced into the SB transposon by using a tetracycline-controlled transrepressor (tTR) protein, consisting of a tetracycline repressor (TetR) fused to the Kruppel-associated box (KRAB) domain of human KOX1 through tetracycline operator (tetO) sequences. The excision frequency of the SB transposon, which is the first step of the transposition event, was enhanced by approximately 100-fold. SB transposase was found to be colocalized with intense DAPI (4',6'-diamidino-2-phenylindole) staining and with the HP1 family by biochemical fractionation analyses. Furthermore, chromatin immunoprecipitation analysis revealed that SB transposase was recruited to tTR-induced heterochromatic regions. These data suggest that the high affinity of SB transposase for heterochromatin conformation leads to enhancement of SB transposition efficiency.     

Sequencing methods and datasets to improve functional interpretation of sleeping beauty mutagenesis screens

Background

Animal models of cancer are useful to generate complementary datasets for comparison to human tumor data. Insertional mutagenesis screens, such as those utilizing the Sleeping Beauty (SB) transposon system, provide a model that recapitulates the spontaneous development and progression of human disease. This approach has been widely used to model a variety of cancers in mice. Comprehensive mutation profiles are generated for individual tumors through amplification of transposon insertion sites followed by high-throughput sequencing. Subsequent statistical analyses identify common insertion sites (CISs), which are predicted to be functionally involved in tumorigenesis. Current methods utilized for SB insertion site analysis have some significant limitations. For one, they do not account for transposon footprints – a class of mutation generated following transposon remobilization. Existing methods also discard quantitative sequence data due to uncertainty regarding the extent to which it accurately reflects mutation abundance within a heterogeneous tumor. Additionally, computational analyses generally assume that all potential insertion sites have an equal probability of being detected under non-selective conditions, an assumption without sufficient relevant data. The goal of our study was to address these potential confounding factors in order to enhance functional interpretation of insertion site data from tumors.

Results

We describe here a novel method to detect footprints generated by transposon remobilization, which revealed minimal evidence of positive selection in tumors. We also present extensive characterization data demonstrating an ability to reproducibly assign semi-quantitative information to individual insertion sites within a tumor sample. Finally, we identify apparent biases for detection of inserted transposons in several genomic regions that may lead to the identification of false positive CISs.

Conclusion

The information we provide can be used to refine analyses of data from insertional mutagenesis screens, improving functional interpretation of results and facilitating the identification of genes important in cancer development and progression.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1150) contains supplementary material, which is available to authorized users.     

Identification of Yersinia enterocolitica genes affecting survival in an animal host using signature-tagged transposon mutagenesis

Pathogenic Yersinia species are associated with both localized and systemic infections in mammalian hosts. In this study, signature-tagged transposon mutagenesis was used to identify Yersinia enterocolitica genes required for survival in a mouse model of infection. Approximately 2000 transposon insertion mutants were screened for attenuation. This led to the identification of 55 mutants defective for survival in the animal host, as judged by their ability to compete with the wild-type strain in mixed infections. A total of 28 mutants had transposon insertions in the virulence plasmid, validating the screen. Two of the plasmid mutants with severe virulence defects had insertions in an uncharacterized region. Several of the chromosomal insertions were in a gene cluster involved in O-antigen biosynthesis. Other chromosomal insertions identified genes not previously demonstrated as being required for in vivo survival of Y. enterocolitica. These include genes involved in the synthesis of outer membrane components, stress response and nutrient acquisition. One severely attenuated mutant had an insertion in a homologue of the pspC gene (phage shock protein C) of Escherichia coli. The phage shock protein operon has no known biochemical or physiological function in E. coli, but is apparently essential for the survival of Y. enterocolitica during infection.     

Functional conservation of the Salmonella and Shigella effectors of entry into epithelial cells

A Salmonella typhi chromosomal locus composed of five adjacent genes, designated sipEBCDA , was identified by transposon mutagenesis as being essential for cell invasion. Products of the sip genes exhibit extensive sequence similarities to the effectors of Shigella entry into epithelial cells encoded by the virulence plasmid-borne ipa operon. Expression of sipE and sipB in a Shigella non-invasive ipaB mutant restored the ability to invade epithelial cells. The structural and functional conservation of the Sip and Ipa proteins suggests that Salmonella and Shigella entry processes are promoted by similar effectors.     

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.