Harnessing a high cargo-capacity transposon for genetic applications in vertebrates

Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrate genomes but exhibit diminished activity when cargo exceeds 8 kilobases (kb). This size restriction limits their molecular genetic and biotechnological utility, such as numerous therapeutically relevant genes that exceed 8 kb in size. Furthermore, a greater payload capacity vector would accommodate more sophisticated cis cargo designs to modulate the expression and mutagenic risk of these molecular therapeutics. We show that the Tol2 transposon can efficiently integrate DNA sequences larger than 10 kb into human cells. We characterize minimal sequences necessary for transposition (miniTol2) in vivo in zebrafish and in vitro in human cells. Both the 8.5-kb Tol2 transposon and 5.8-kb miniTol2 engineered elements readily function to revert the deficiency of fumarylacetoacetate hydrolase in an animal model of hereditary tyrosinemia type 1. Together, Tol2 provides a novel nonviral vector for the delivery of large genetic payloads for gene therapy and other transgenic applications.     

The <Emphasis Type="Italic">piggyBac</Emphasis> Transposon as a Tool in Genetic Engineering

Transposons are mobile genetic elements that are part of the genomic DNA of numerous organisms and belong to two classes. Unlike class I transposons, class II DNA transposons do not use the stage of RNA synthesis in their transition; they perform it by the cut-and-paste mechanism or with a replicative transposition. The integration of a DNA transposon in a new site results in the duplication of a target sequence on either side of a transposon, and its excision is, as a rule, associated with insertions and deletions. The piggyBac transposon isolated from the Trichoplusia ni moth differs from other mobile elements of its class. Due to its unique ability to leave no traces after excision from an insertion site and to perform successful transposition and transference of large DNA fragments, piggyBac is a convenient tool for the development of gene engineering approaches. The TTAA sequence serves as a target site for transposon integration: insertion in the AT-rich DNA regions is more frequent. The ability of piggyBac to be transferred to a new area independently of the cell apparatus and to restore a DNA site without error after excision lies in the mechanism of its transposition, which is discussed in detail in the present review. Along with other transposons and viruses, the piggyBac transposon is widely used in the transgenesis of various organisms; it also finds application in insertion mutagenesis and gene therapy.     

转座子PiggyBac在哺乳动物中的应用

DNA转座子作为一种遗传工程工具已广泛应用于多物种的转基因及产生插入突变等研究。目前,在哺乳动物中有转座活性的转座子可分为三类：1）hAT样转座子;2）Tcl样转座子包括Sleeping Beauty和FrogPrince;3）PiggyBac转座子家族。其中甘蓝蠖度尺蛾（Cabbage looper moth Trichoplusia ni）来源的PiggyBac转座子是目前在哺乳动物中活性最高的转座子,并且可以携带十几kb的外源基因转座而不影响其效率,使其在哺乳动物的转基因、癌基因的发现、基因治疗研究方面具有巨大的应用潜力。此外,PB的无痕迹转座对于无转基因、无遗传物质改变的诱导多潜能干细胞（iPS）研究也具有非常重要的意义。本文主要对针对PB在哺乳动物中的应用现状及前景作一介绍。     

Tdr2, a new zebrafish transposon of the Tc1 family.

We describe here Tdr2, a new class of Tc1-like transposons in zebrafish. Tdr2 was identified from the genomic sequence of a zebrafish PAC (P1 artificial chromosome) clone, and fragments of Tdr2 were found in several zebrafish EST (expressed sequence tag) sequences. Predicted translation of the Tdr2 transposase gene showed that it was most closely related to Caenorhabditis elegans Tc3A, suggesting an ancient origin of the Tdr2 transposon. Tdr2 spans 1. 1kb and is flanked by inverted repeats of approx. 100bp. The 5' repeat is itself composed of an inverted repeat, raising the possibility of the formation of a cruciform DNA structure. Tdr2 transposons may facilitate the development of novel transposon-based tools for the genetic analysis of zebrafish.     

转座子引起的猪ktn1基因结构变异及其与生产性能的关联分析

为探明转座子对猪的ktn1基因及其侧翼区结构变异的贡献,从全基因组测序(WGS)数据库中获取14个猪基因组中的ktn1基因序列和侧翼序列,通过ClustalX多序列比对和RepeatMasker转座子注释,全面解析转座子对ktn1的影响。通过PCR检测到一个SINEA1转座子插入多态,在苏姜猪群体中与相关性状进行关联分析。结果显示,ktn1基因及其侧翼区中含有至少77个转座子片段,其中绝大部分(98.70%)为SINE类转座子,并鉴定到9个小结构变异和4个由转座子引起的大结构变异,表明转座子是基因变异的重要来源。其中一个SINEA1插入多态引起的结构变异,在不同品种中呈现丰富的多态性,且无插入个体(SINE-/-)苏姜猪的断奶窝重((64.20±10.6) kg)比纯合有插入个体(SINE+/+)((74.14±9.0) kg)和杂合有插入个体(SINE+/-)((69.71±7.7) kg)轻(P<0.05),表明基于转座子插入多态研发分子标记具有可行性,提示转座子插入多态分子标记在分子辅助育种中具有较强的应用潜力。     

水稻转座子研究进展

转座子是植物基因组的重要组成部分, 对于研究植物基因组进化等具有重要意义。随着水稻全基因组测序计划的开展和完成, 水稻转座子研究取得了极大进展, 目前已经在水稻基因组中发现了几乎所有类型的转座子, 约占水稻基因组的35%。在正常情况下, 大多数水稻转座子不具有转座活性, 但是在特定的条件下(如组织培养或辐射等), 水稻基因组中沉默的转座子可以被激活, 从而可能导致插入突变并影响基因的表达。在水稻中已鉴定出6个有活性的转座子, 其中Tos17已被应用到水稻功能基因组研究中。转座子序列的新的分子标记转座子展示(transposon display, TD)现已被开发, 并在水稻遗传作图和遗传分化研究中得到应用     

piggyBac转座系统的功能改进及在哺乳动物中的应用

piggyBac (PB)转座系统具有转座效率高、删除精确、半随机插入和携带片段较大等优点。但是作为一种转基因实验的工具,特别是在哺乳动物个体水平的转基因方面,还需要提高其转基因效率,并降低外源基因随机插入对内源基因破坏的风险。近年来的研究结果显示,PB转座系统得到了进一步改进：采用PB转座酶与DNA特异性结合蛋白融合而构成的融合型转座酶,表现出外源片段有插入到染色体靶向位点的倾向;采用突变体筛选的方法提高了PB转座酶的活性,获得了只具有切除活性而没有插入活性的新型PB转座酶;采用PB转座系统与细菌人工染色体(Bacterial artificial chromosomes, BAC)载体联合使携带的外源片段长度提高到了207 kb。改进后的PB转座系统在基因组研究、基因治疗、诱导多能干细胞(Induced pluripotent stem cells, iPSCs)诱导及其分化方面发挥了较大的作用。文章对PB转座系统的最新研究进展和应用前景进行了综述。     

Global mapping of transposon location

Transposable genetic elements are ubiquitous, yet their presence or absence at any given position within a genome can vary between individual cells, tissues, or strains. Transposable elements have profound impacts on host genomes by altering gene expression, assisting in genomic rearrangements, causing insertional mutations, and serving as sources of phenotypic variation. Characterizing a genome's full complement of transposons requires whole genome sequencing, precluding simple studies of the impact of transposition on interindividual variation. Here, we describe a global mapping approach for identifying transposon locations in any genome, using a combination of transposon-specific DNA extraction and microarray-based comparative hybridization analysis. We use this approach to map the repertoire of endogenous transposons in different laboratory strains of Saccharomyces cerevisiae and demonstrate that transposons are a source of extensive genomic variation. We also apply this method to mapping bacterial transposon insertion sites in a yeast genomic library. This unique whole genome view of transposon location will facilitate our exploration of transposon dynamics, as well as defining bases for individual differences and adaptive potential.     

Epigenetic changes and transposon reactivation in Thai rice hybrids

Inter- or intraspecific hybridization is the first step in transferring exogenous traits to the germplasm of a recipient crop. One of the complicating factors is the occurrence of epigenetic modifications of the hybrids, which in turn can change their gene expression and phenotype. In this study we present an analysis of epigenome changes in rice hybrids that were obtained by crossing rice cultivars, most of them of indica type and Thai origin. Comparing amplified fragment length polymorphism (AFLP) fingerprints of twenty-four cultivars, we calculated Nei’s indexes for measuring genetic relationships. Epigenetic changes in their hybrids were established using methylation-sensitive AFLP fingerprinting and transposon display of the rice transposable elements (TEs) Stowaway Os-1 and Mashu, leading to the question whether the relationship between parental genomes is a predictor of epigenome changes, TE reactivation and changes in TE methylation. Our study now reveals that the genetic relationship between the parents and DNA methylation changes in their hybrids is not significantly correlated. Moreover, genetic distance correlates only weakly with Mashu reactivation, whereas it does not correlate with Stowaway Os-1 reactivation. Our observations also suggest that epigenome changes in the hybrids are localized events affecting specific chromosomal regions and transposons rather than affecting the genomic methylation landscape as a whole. The weak correlation between genetic distance and Mashu methylation and reactivation points at only limited influence of genetic background on the epigenetic status of the transposon. Our study further demonstrates that hybridizations between and among specific japonica and indica cultivars induce both genomic DNA methylation and reactivation/methylation change in the Stowaway Os-1 and Mashu transposons. The observed epigenetic changes seem to affect the transposons in a clear manner, partly driven by stochastic processes, which may account for a broader phenotypic plasticity of the hybrids. A better understanding of the epigenome changes leading to such transposon activation can lead to the development of novel tools for more variability in future rice breeding.     

Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells

Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns. We evaluated if there were undesired genomic effects of the PB transposon system to modify human cells. Expression of the transposase alone revealed no mobilization of endogenous PB-like sequences in the human genome and no increase in DNA double-strand breaks. The use of PB in a plasmid containing both transposase and transposon greatly increased the probability of transposase integration; however, using transposon and transposase from separate vectors circumvented this. Placing a eGFP transgene within transposon vector backbone allowed isolation of cells free from vector backbone DNA. We confirmed observable directional promoter activity within the 5′TR element of PB but found no significant enhancer effects from the transposon DNA sequence. Long-term culture of primary human cells modified with eGFP-transposons revealed no selective growth advantage of transposon-harboring cells. PB represents a promising vector system for genetic modification of human cells with limited undesired genomic effects.     

An active DNA transposon nDart causing leaf variegation and mutable dwarfism and its related elements in rice

While characterized mutable alleles caused by DNA transposons have been abundant in maize since the discovery of Dissociation conferring variegation by Barbara McClintock, only a few mutable alleles have been described in rice even though the rice genome contains various transposons. Here, we show that a spontaneous mutable virescent allele, pyl-v, is caused by the disruption of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to insertion of a 607-bp non-autonomous DNA transposon, non-autonomous DNA-based active rice transposon one (nDart1), belonging to the hAT superfamily. The transposition of nDart1 can be induced by crossing with a line containing an autonomous element, aDart, and stabilized by segregating out of aDart. We also identified a novel mutable dwarf allele thl-m caused by an insertion of nDart1. The japonica cultivar Nipponbare carries no aDart, although it contains epigenetically silenced Dart element(s), which can be activated by 5-azacytidine. Nipponbare bears four subgroups of about 3.6-kb Dart-like sequences, three of which contain potential transposase genes, and around 3.6-kb elements without an apparent transposase gene, as well as three subgroups of about 0.6-kb nDart1-related elements that are all internal deletions of the Dart-like sequences. Both nDart1 and 3.6-kb Dart-like elements were also present in indica varieties 93-11 and Kasalath. nDart1 appears to be the most active mutagen among nDart1-related elements contributing to generating natural variations. A candidate for an autonomous element, aDart, and a possible application of nDart1 for transposon tagging are discussed.     

Identification of the Maize Gravitropism Gene lazy plant1 by a Transposon-Tagging Genome Resequencing Strategy

Since their initial discovery, transposons have been widely used as mutagens for forward and reverse genetic screens in a range of organisms. The problems of high copy number and sequence divergence among related transposons have often limited the efficiency at which tagged genes can be identified. A method was developed to identity the locations of Mutator (Mu) transposons in the Zea mays genome using a simple enrichment method combined with genome resequencing to identify transposon junction fragments. The sequencing library was prepared from genomic DNA by digesting with a restriction enzyme that cuts within a perfectly conserved motif of the Mu terminal inverted repeats (TIR). Paired-end reads containing Mu TIR sequences were computationally identified and chromosomal sequences flanking the transposon were mapped to the maize reference genome. This method has been used to identify Mu insertions in a number of alleles and to isolate the previously unidentified lazy plant1 (la1) gene. The la1 gene is required for the negatively gravitropic response of shoots and mutant plants lack the ability to sense gravity. Using bioinformatic and fluorescence microscopy approaches, we show that the la1 gene encodes a cell membrane and nuclear localized protein. Our Mu-Taq method is readily adaptable to identify the genomic locations of any insertion of a known sequence in any organism using any sequencing platform.     

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.     

Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis.

We have developed efficient methods for creating artificial transposons and inserting these transposons into plasmid targets in vitro, primarily for the purpose of DNA mapping and sequencing. A novel plasmid has been engineered to convert virtually any DNA sequence, or combination of sequences, into an artificial transposon; hence, custom transposons containing any desired feature can be easily designed and constructed. Such transposons are then efficiently inserted into plasmid targets, in vitro, using the integrase activity present in yeast Ty1 virus-like particles. A single in vitro integration reaction, which resembles a simple restriction digestion in the complexity of the reaction, gives rise to thousands of recoverable insertion events within DNA target molecules; this frequency approaches one insertion per phosphodiester bond in typical plasmids. Importantly, transposon insertions are recovered from all regions of DNA inserts carried on plasmid targets, indicating that integration is a random or nearly-random process. Because of its versatility, this technology offers a generalized method of generating recombinant DNA molecules of a desired structure. We have adapted this system for DNA sequencing by developing a customized artificial transposon to insert new primer binding sites into internal regions of DNA inserts carried on cloning vectors. Transposon insertions have been generated throughout several different yeast and human DNA inserts carried on plasmids, allowing the efficient recovery of sequence information from these inserts. Our results demonstrate the overall utility of this method for both small and large-scale DNA sequencing, as well as general DNA restructuring, and indicate that it could be adapted for use with a number of additional applications including functional genetic analysis.     

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.     

Minimization of the Escherichia coli genome using a Tn5-targeted Cre/loxP excision system

An increasing number of microbial genomes have been completely sequenced, and functional analyses of these genomic sequences are under way. To facilitate these analyses, we have developed a genome-engineering tool for determining essential genes and minimizing bacterial genomes. We made two large pools of independent transposon mutants in Escherichia coli using modified Tn5 transposons with two different selection markers and precisely mapped the chromosomal location of 800 of these transposons. By combining a mapped transposon mutation from each of the mutant pools into the same chromosome using phage P1 transduction and then excising the flanked genomic segment by Cre-mediated loxP recombination, we obtained E. coli strains in which large genomic fragments (59-117 kilobases) were deleted. Some of these individual deletions were then combined into a single "cumulative deletion strain" that lacked 287 open reading frames (313.1 kilobases) but that nevertheless exhibited normal growth under standard laboratory conditions.     

Structural features of 3C6/C7 interband chromatin organization in Drosophila melanogaster polytene chromosomes

Mechanisms of chromatin decompaction in interbands of Drosophila polytene chromosomes have been studied. Using the example of interband 3C6/C7 of the X chromosome, we investigated the ability of different DNA segments to form an interband in a new genetic environment. We applied site-specific FLP recombination between two transposons with FRT-sites that allows introducing the DNA fragments from the interband 3C6/C7 into pICon(dv) transposon located in cytologically well-characterized 84F region of chromosome 3 followed by electron microscopic analysis of changes in the region caused by insertion of the DNA fragments into the transposon. It was shown that the insertion of a 276-bp DNA fragment from the 3C6/C7 region into the pICon(dv) transposon leads to the formation of a new interband between two thin bands represented by the transposon material. This DNA fragment is the known minimal sequence that is necessary and sufficient for interband generation. In addition, the sequence containing three copies repeated in tandem of 0.9 kb DNA from the interband 3C6/C7, including the 276-bp fragment, were integrated in the transposon. The presence of introduced DNA fragments did not change the morphology of the resulting interband. It was shown that the sites of DNase I hypersensitivity were saved in transposon sequences introduced into a new genetic environment. The data obtained allow analysis to be started of specific factors (proteins, DNA motifs, etc.) that determine the formation of decompacted chromatin in a certain interband region and chromomeric organization of interphase chromosomes in Drosophila as a whole.     

DNA transposons: nature and applications in genomics

Repeated DNA makes up a large fraction of a typical mammalian genome, and some repetitive elements are able to move within the genome (transposons and retrotransposons). DNA transposons move from one genomic location to another by a cut-and-paste mechanism. They are powerful forces of genetic change and have played a significant role in the evolution of many genomes. As genetic tools, DNA transposons can be used to introduce a piece of foreign DNA into a genome. Indeed, they have been used for transgenesis and insertional mutagenesis in different organisms, since these elements are not generally dependent on host factors to mediate their mobility. Thus, DNA transposons are useful tools to analyze the regulatory genome, study embryonic development, identify genes and pathways implicated in disease or pathogenesis of pathogens, and even contribute to gene therapy. In this review, we will describe the nature of these elements and discuss recent advances in this field of research, as well as our evolving knowledge of the DNA transposons most widely used in these studies.