Efficient mammalian germline transgenesis by cis-enhanced <Emphasis Type="Italic">Sleeping Beauty</Emphasis> transposition

Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline.     

Chromosomal mobilization and reintegration of Sleeping Beauty and PiggyBac transposons

The Sleeping Beauty and PiggyBac DNA transposon systems have recently been developed as tools for insertional mutagenesis. We have compared the chromosomal mobilization efficiency and insertion site preference of the two transposons mobilized from the same donor site in mouse embryonic stem (ES) cells under conditions in which there were no selective constraints on the transposons' insertion sites. Compared with Sleeping Beauty, PiggyBac exhibits higher transposition efficiencies, no evidence for local hopping and a significant bias toward reintegration in intragenic regions, which demonstrate its utility for insertional mutagenesis. Although Sleeping Beauty had no detectable genomic bias with respect to insertions in genes or intergenic regions, both Sleeping Beauty and PiggyBac transposons displayed preferential integration into actively transcribed loci. genesis 47:404–408, 2009. © 2009 Wiley‐Liss, Inc.     

Translating Sleeping Beauty transposition into cellular therapies: Victories and challenges

Recent results confirm that long‐term expression of therapeutic transgenes can be achieved by using a transposon‐based system in primary stem cells and in vivo. Transposable elements are natural DNA transfer vehicles that are capable of efficient genomic insertion. The latest generation, Sleeping Beauty transposon‐based hyperactive vector (SB100X), is able to address the basic problem of non‐viral approaches – that is, low efficiency of stable gene transfer. The combination of transposon‐based non‐viral gene transfer with the latest improvements of non‐viral delivery techniques could provide a long‐term therapeutic effect without compromising biosafety. The new challenges of pre‐clinical research will focus on further refinement of the technology in large animal models and improving the safety profile of SB vectors by target‐selected transgene integration into genomic “safe harbors.” The first clinical application of the SB system will help to validate the safety of this approach.     

Generation of rat mutants using a coat color-tagged <Emphasis Type="Italic">Sleeping Beauty</Emphasis> transposon system

A significant barrier to exploiting the full potential of the rat as a biomedical model is the lack of tools to easily modify its germline. Here we show that a tyrosinase-tagged Sleeping Beauty transposon can be used as a simple, efficient method to generate rat mutants in vivo. By making two lines of transgenic rats, one carrying the transposon and another expressing the transposase in germ cells, we are able to obtain bigenic males in which transposition occurs in the germ cells. We show that transposition leads to the appearance of new coat colors in the offspring. Using such bigenic males, we obtained an average of 1.2 transpositions per gamete and identified 19 intragenic integration events among 96 transposition sites that were sequenced. In addition, gene trapping was confirmed and rats with evidence for transposon-induced dominant ocular anomalies were identified. These data suggest that the modified Sleeping Beauty transposon represents a powerful new tool for producing molecularly defined mutagenesis in the rat.     

Genetic Signature of Histiocytic Sarcoma Revealed by a Sleeping Beauty Transposon Genetic Screen in Mice

Histiocytic sarcoma is a rare, aggressive neoplasm that responds poorly to therapy. Histiocytic sarcoma is thought to arise from macrophage precursor cells via genetic changes that are largely undefined. To improve our understanding of the etiology of histiocytic sarcoma we conducted a forward genetic screen in mice using the Sleeping Beauty transposon as a mutagen to identify genetic drivers of histiocytic sarcoma. Sleeping Beauty mutagenesis was targeted to myeloid lineage cells using the Lysozyme2 promoter. Mice with activated Sleeping Beauty mutagenesis had significantly shortened lifespan and the majority of these mice developed tumors resembling human histiocytic sarcoma. Analysis of transposon insertions identified 27 common insertion sites containing 28 candidate cancer genes. Several of these genes are known drivers of hematological neoplasms, like Raf1, Fli1, and Mitf, while others are well-known cancer genes, including Nf1, Myc, Jak2, and Pten. Importantly, several new potential drivers of histiocytic sarcoma were identified and could serve as targets for therapy for histiocytic sarcoma patients.     

Generating mutant rats using the Sleeping Beauty transposon system

The laboratory rat is an invaluable animal model for biomedical research. However, mutant rat resource is still limited, and development of methods for large-scale generation of mutants is anticipated. We recently utilized the Sleeping Beauty (SB) transposon system to develop a rapid method for generating insertional mutant rats. Firstly, transgenic rats carrying single transgenes, namely the SB transposon vector and SB transposase, were generated. Secondly, these single transgenic rats were interbred to obtain doubly-transgenic rats carrying both transgenes. The SB transposon was mobilized in the germline of these doubly-transgenic rats, reinserted into another location in the genome and heterozygous mutant rats were obtained in the progeny. Gene insertion events were rapidly and non-invasively identified by the green fluorescence protein (GFP) reporter incorporated in the transposon vector, which utilizes a polyA-trap approach. Mutated genes were confirmed by either linker ligation-mediated PCR or 3′-rapid amplification of cDNA ends (3′RACE). Endogenous expression profile of the mutated gene can also be visualized using the LacZ gene incorporated as a promoter-trap unit in the transposon vector. This method is straightforward, readily applicable to other transposon systems, and will be a valuable mutant rat resource to the biomedical research community.     

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.     

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.     

Sleeping Beauty transposition: from biology to applications

Sleeping Beauty (SB) is the first synthetic DNA transposon that was shown to be active in a wide variety of species. Here, we review studies from the last two decades addressing both basic biology and applications of this transposon. We discuss how host–transposon interaction modulates transposition at different steps of the transposition reaction. We also discuss how the transposon was translated for gene delivery and gene discovery purposes. We critically review the system in clinical, pre-clinical and non-clinical settings as a non-viral gene delivery tool in comparison with viral technologies. We also discuss emerging SB-based hybrid vectors aimed at combining the attractive safety features of the transposon with effective viral delivery. The success of the SB-based technology can be fundamentally attributed to being able to insert fairly randomly into genomic regions that allow stable long-term expression of the delivered transgene cassette. SB has emerged as an efficient and economical toolkit for safe and efficient gene delivery for medical applications.     

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.     

Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system

Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Genomic Analysis of Sleeping Beauty Transposon Integration in Human Somatic Cells

The Sleeping Beauty (SB) transposon is a non-viral integrating vector system with proven efficacy for gene transfer and functional genomics. However, integration efficiency is negatively affected by the length of the transposon. To optimize the SB transposon machinery, the inverted repeats and the transposase gene underwent several modifications, resulting in the generation of the hyperactive SB100X transposase and of the high-capacity “sandwich” (SA) transposon. In this study, we report a side-by-side comparison of the SA and the widely used T2 arrangement of transposon vectors carrying increasing DNA cargoes, up to 18 kb. Clonal analysis of SA integrants in human epithelial cells and in immortalized keratinocytes demonstrates stability and integrity of the transposon independently from the cargo size and copy number-dependent expression of the cargo cassette. A genome-wide analysis of unambiguously mapped SA integrations in keratinocytes showed an almost random distribution, with an overrepresentation in repetitive elements (satellite, LINE and small RNAs) compared to a library representing insertions of the first-generation transposon vector and to gammaretroviral and lentiviral libraries. The SA transposon/SB100X integrating system therefore shows important features as a system for delivering large gene constructs for gene therapy applications.     

Effects of Insert Size on Transposition Efficiency of the Sleeping Beauty Transposon in Mouse Cells

Transposon vectors are widely used in prokaryotic and lower eukaryotic systems. However, they were not available for use in vertebrate animals until the recent reconstitution of a synthetic fish transposon, Sleeping Beauty (SB). The reacquisition of transposability of the SB transposase fostered great enthusiasm for using transposon vectors as tools in vertebrate animals, particularly for gene transfer to facilitate accelerated integration of transgenes into chromosomes. Here, we report the effects of insert sizes on transposition efficiency of SB. A significant effect of insert size on efficiency of transposition by SB was found. The SB transposase enhanced the integration efficiency effectively for SB transposon up to approximately 5.6 kb, but lost its ability to enhance the integration efficiency when the transposon size was increased to 9.1 kb. This result indicates that the SB transposon system is highly applicable for transferring small genes, but may not be applicable for transferring very large genes. Received October 20, 2000; accepted December 15, 2000.     

Efficient stable gene transfer into human cells by the Sleeping Beauty transposon vectors

Transposable elements can be considered as natural, non-viral gene delivery vehicles capable of efficient genomic insertion. The plasmid-based transposon system of Sleeping Beauty (SB) combines the advantages of viruses and naked DNA molecules. In contrast to plasmid vectors, transposons integrate through a precise, recombinase-mediated mechanism into chromosomes, providing long-term expression of the gene of interest in cells. The advantages of transposons in comparison to viral systems include their simplicity and improved safety/toxicity profiles. In addition, the hyperactive SB100X is the first plasmid-based delivery system that overcomes the efficacy of non-viral delivery. The transposon delivery system consists of the transposase and the integration cassette, recognized by the transposase. The plasmid-based transposon delivery system can be combined with any non-viral delivery method. Here we provide two detailed protocols to apply SB-mediated, non-viral gene transfer in cultured cells. In our first example, we use a lipid-based delivery method in combination with the transposon-based integration system in an easy-to-transfect (HeLa) cell line. Second, we show how to achieve 40–50% stable expression of a transgene in clinically relevant, hard-to-transfect cells (hematopoetic stem cells, HSCs) by nucleofection. The given protocols are adaptable to any vertebrate cells in culture.     

NMR structural analysis of Sleeping Beauty transposase binding to DNA

The Sleeping Beauty (SB) transposon is the most widely used DNA transposon in genetic applications and is the only DNA transposon thus far in clinical trials for human gene therapy. In the absence of atomic level structural information, the development of SB transposon relied primarily on the biochemical and genetic homology data. While these studies were successful and have yielded hyperactive transposases, structural information is needed to gain a mechanistic understanding of transposase activity and guides to further improvement. We have initiated a structural study of SB transposase using Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) spectroscopy to investigate the properties of the DNA‐binding domain of SB transposase in solution. We show that at physiologic salt concentrations, the SB DNA‐binding domain remains mostly unstructured but its N‐terminal PAI subdomain forms a compact, three‐helical structure with a helix‐turn‐helix motif at higher concentrations of NaCl. Furthermore, we show that the full‐length SB DNA‐binding domain associates differently with inner and outer binding sites of the transposon DNA. We also show that the PAI subdomain of SB DNA‐binding domain has a dominant role in transposase's attachment to the inverted terminal repeats of the transposon DNA. Overall, our data validate several earlier predictions and provide new insights on how SB transposase recognizes transposon DNA.     

DNA transposon-based gene vehicles - scenes from an evolutionary drive

DNA transposons are primitive genetic elements which have colonized living organisms from plants to bacteria and mammals. Through evolution such parasitic elements have shaped their host genomes by replicating and relocating between chromosomal loci in processes catalyzed by the transposase proteins encoded by the elements themselves. DNA transposable elements are constantly adapting to life in the genome, and self-suppressive regulation as well as defensive host mechanisms may assist in buffering ‘cut-and-paste’ DNA mobilization until accumulating mutations will eventually restrict events of transposition. With the reconstructed Sleeping Beauty DNA transposon as a powerful engine, a growing list of transposable elements with activity in human cells have moved into biomedical experimentation and preclinical therapy as versatile vehicles for delivery and genomic insertion of transgenes. In this review, we aim to link the mechanisms that drive transposon evolution with the realities and potential challenges we are facing when adapting DNA transposons for gene transfer. We argue that DNA transposon-derived vectors may carry inherent, and potentially limiting, traits of their mother elements. By understanding in detail the evolutionary journey of transposons, from host colonization to element multiplication and inactivation, we may better exploit the potential of distinct transposable elements. Hence, parallel efforts to investigate and develop distinct, but potent, transposon-based vector systems will benefit the broad applications of gene transfer. Insight and clever optimization have shaped new DNA transposon vectors, which recently debuted in the first DNA transposon-based clinical trial. Learning from an evolutionary drive may help us create gene vehicles that are safer, more efficient, and less prone for suppression and inactivation.     

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.     

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.     

Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells

The discovery of direct cell reprogramming and induced pluripotent stem (iPS) cell technology opened up new avenues for the application of non-viral, transposon-based gene delivery systems. The Sleeping Beauty (SB) transposon is highly advanced for versatile genetic manipulations in mammalian cells. We established iPS cell reprogramming of mouse embryonic fibroblasts and human foreskin fibroblasts by transposition of OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28) expression cassettes mobilized by the SB100X hyperactive transposase. The efficiency of iPS cell derivation with SB transposon system was in the range of that obtained with retroviral vectors. Co-expression of the miRNA302/367 cluster together with OSKM significantly improved reprogramming efficiency and accelerated the temporal kinetics of reprogramming. The iPS cells displayed a stable karyotype, and hallmarks of pluripotency including expression of stem cell markers and the ability to differentiate into embryoid bodies in vitro. We demonstrate Cre recombinase-mediated exchange allowing simultaneous removal of the reprogramming cassette and targeted knock-in of an expression cassette of interest into the transposon-tagged locus in mouse iPS cells. This strategy would allow correction of a genetic defect by site-specific insertion of a therapeutic gene construct into ‘safe harbor’ sites in the genomes of autologous, patient-derived iPS cells.