Mobilization of giant piggyBac transposons in the mouse genome

The development of technologies that allow the stable delivery of large genomic DNA fragments in mammalian systems is important for genetic studies as well as for applications in gene therapy. DNA transposons have emerged as flexible and efficient molecular vehicles to mediate stable cargo transfer. However, the ability to carry DNA fragments >10 kb is limited in most DNA transposons. Here, we show that the DNA transposon piggyBac can mobilize 100-kb DNA fragments in mouse embryonic stem (ES) cells, making it the only known transposon with such a large cargo capacity. The integrity of the cargo is maintained during transposition, the copy number can be controlled and the inserted giant transposons express the genomic cargo. Furthermore, these 100-kb transposons can also be excised from the genome without leaving a footprint. The development of piggyBac as a large cargo vector will facilitate a wider range of genetic and genomic applications.     

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.     

Chimeric piggyBac transposases for genomic targeting in human cells

Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4-PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24% of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy.     

转座子Sleeping Beauty和PiggyBac

近10年来,得益于转座子Sleeping Beauty(SB)和PiggyBac(PB)的发现和完善,转座子作为一种遗传工程工具在脊椎动物的基因遗传研究中得到广泛应用.SB和PB宿主范围极其广泛,从单细胞生物到哺乳动物都能够发挥作用.转座过程需要转座序列和转座酶的存在,类似于"剪切"、"粘贴"的方式.转座子载体系统转座时可携带一段外源DNA序列,利用这一特性可以用于实现目的基因的转移,现已广泛用于转基因动物、基因功能研究、基因治疗等领域.当转座系统与基因捕获技术相结合,不仅可研究插入突变基因的功能,还能通过所携带的报告基因获得捕获基因的表达图谱.作为非病毒载体的SB和PB转座系统,由于具有高容量、高效率和高安全性等优势,并且PB在转座后不留任何足迹,不会造成遗传物质的不可预测改变,在动物基因工程以及基因治疗方面具有诱人的前景.     

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。     

新型二合一PiggyBac转座系统的构建及功能验证*

目的:PiggyBac(PB)转座子是一种可移动的遗传元件,采用“剪切和粘贴”机制在载体和染色体之间进行转座;通过将转座子元件和转座酶表达框整合到一个表达载体中,构建简便易用的二合一PB转座系统。方法:通过聚合酶链式反应(polymerase chain reaction,PCR)获取PiggyBac转座系统所需转座子元件和转座酶表达框,利用T4 DNA连接酶将转座酶表达框插入到pUC18载体上,再利用Gibson同源重组技术将转座子元件与重组载体结合构建二合一PB转座系统;使用该系统携带的增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)以及功能性损伤抑制蛋白(damage-suppressing protein,DSUP)检测其有效性及可靠性。结果:在所有筛选获得的嘌呤霉素抗性细胞中,EGFP都是明亮可见;利用此二合一PB转座系统成功获得了可高效表达功能性损伤抑制蛋白的稳定细胞系,证明外源基因可被有效整合到基因组DNA中并表达。结论:成功构建了新型二合一PB转座系统,使稳定表达细胞系的建立更加经济简便。     

Optimization of the piggyBac Transposon Using mRNA and Insulators: Toward a More Reliable Gene Delivery System

Integrating and expressing stably a transgene into the cellular genome remain major challenges for gene-based therapies and for bioproduction purposes. While transposon vectors mediate efficient transgene integration, expression may be limited by epigenetic silencing, and persistent transposase expression may mediate multiple transposition cycles. Here, we evaluated the delivery of the piggyBac transposase messenger RNA combined with genetically insulated transposons to isolate the transgene from neighboring regulatory elements and stabilize expression. A comparison of piggyBac transposase expression from messenger RNA and DNA vectors was carried out in terms of expression levels, transposition efficiency, transgene expression and genotoxic effects, in order to calibrate and secure the transposition-based delivery system. Messenger RNA reduced the persistence of the transposase to a narrow window, thus decreasing side effects such as superfluous genomic DNA cleavage. Both the CTF/NF1 and the D4Z4 insulators were found to mediate more efficient expression from a few transposition events. We conclude that the use of engineered piggyBac transposase mRNA and insulated transposons offer promising ways of improving the quality of the integration process and sustaining the expression of transposon vectors.     

piggyBac转座子在牛基因组的整合位点及特征分析

piggyBac(PB)转座子作为一种遗传工具被广泛应用于多个物种的转基因及插入突变研究, 目前PB转座子在牛中的相关研究还较少。为了获得PB转座子在牛基因组中的整合位点, 总结其转座特征, 文章构建了PB[CMV-EGFP]和pcDNA-PBase二元转座系统, 利用细胞核电转技术共转染牛耳组织成纤维细胞, 经G-418筛选, 获得了稳定转染EGFP的转基因细胞系; 提取细胞基因组DNA, 利用基因组步移技术扩增PB转座子5′ Bac区插入位置的DNA序列; 通过与牛基因组序列进行BLAST比对, 得到PB转座子在牛基因组中的插入位点。文章共获得了8个有效的整合位点, 但仅有5个位点定位到染色体1、2、11和X染色体上。序列分析表明：在牛基因组中, PB转座子可特异性的插入到“TTAA”位置, 并整合到基因间的非调控区; 分析整合位点“TTAA”相邻一侧的5个碱基组成, 发现PB转座子5′端倾向于插入到GC(62.5%)碱基富集区。该研究表明, PB转座子可以在牛基因组中发生转座, 获得的整合位点信息为利用PB转座子在牛上开展遗传学研究提供了理论参考。     

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.     

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。     

Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells

The Tol2 transposable element of the Japanese medaka fish belongs to the hAT family of transposons including hobo of Drosophila, Ac of maize, and Tam3 of snapdragon. To date, Tol2 is the only natural transposon in vertebrates that has ever been shown to encode a fully functional transposase. It has not been known, however, whether Tol2 can transpose in vertebrates other than fish. We report here transposition of Tol2 in mouse embryonic stem (ES) cells. We constructed a transposon donor plasmid containing a nonautonomous Tol2 element with the neomycin resistance gene and a helper plasmid capable of expressing the transposase and introduced the donor plasmid with various amounts of the helper plasmid by electroporation into mouse ES cells. The number of G418-resistant ES colonies increased as the amount of helper plasmid was increased, in a dose-dependent manner, indicating that the transposase activity elevated the integration efficiency. These G418-resistant ES colonies were cloned and the structure of the junction of the integrated Tol2 element and the genomic DNA was analyzed by inverse PCR. In those clones, Tol2 was surrounded by mouse genomic sequences and an 8-bp direct repeat was created adjacent to both ends of Tol2, indicating that Tol2 was integrated in the genome through transposition. The Tol2 transposon system is thus active in mouse as well as in fish. We propose that it should be used as a genetic tool to develop novel gene transfer, transgenesis, and mutagenesis methods in mammals.     

Horizontal Escape of the Novel Tc1-Like Lepidopteran Transposon TCp3.2 into Cydia pomonella Granulovirus

We characterized an insertion mutant of the baculovirus Cydia pomonella granulovirus (CpGV), which contained a transposable element of 3.2 kb. This transposon, termed TCp3.2, has unusually long inverted terminal repeats (ITRs) of 756 bp and encodes a defective gene for a putative transposase. Amino acid sequence comparison of the defective transposase gene revealed a distant relationship to a putative transposon in Caenorhabditis elegans which also shares some similarity of the ITRs. Maximum parsimony analysis of the predicted amino acid sequences of Tc1- and mariner-like transposases available from the GenBank data base grouped TCp3.2 within the superfamily of Tc1-like transposons. DNA hybridization indicated that TCp3.2 originated from the genome of Cydia pomonella, which is the natural host of CpGV, and is present in less than 10 copies in the C. pomonella genome. The transposon TCp3.2 most likely was inserted into the viral genome during infection of host larvae. TCp3.2 and the recently characterized Tc1-like transposon TC14.7 (Jehle et al. 1995), which was also found in a CpGV mutant, represent a new family of transposons found in baculovirus genomes. The occasional horizontal escape of different types of host transposons into baculovirus genomes evokes the question about the possible role of baculoviruses as an interspecies vector in the horizontal transmission of insect transposons. Received: 27 February 1997 / Accepted: 16 May 1997     

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.     

Solution conformations of early intermediates in Mos1 transposition

DNA transposases facilitate genome rearrangements by moving DNA transposons around and between genomes by a cut-and-paste mechanism. DNA transposition proceeds in an ordered series of nucleoprotein complexes that coordinate pairing and cleavage of the transposon ends and integration of the cleaved ends at a new genomic site. Transposition is initiated by transposase recognition of the inverted repeat sequences marking each transposon end. Using a combination of solution scattering and biochemical techniques, we have determined the solution conformations and stoichiometries of DNA-free Mos1 transposase and of the transposase bound to a single transposon end. We show that Mos1 transposase is an elongated homodimer in the absence of DNA and that the N-terminal 55 residues, containing the first helix-turn-helix motif, are required for dimerization. This arrangement is remarkably different from the compact, crossed architecture of the dimer in the Mos1 paired-end complex (PEC). The transposase remains elongated when bound to a single-transposon end in a pre-cleavage complex, and the DNA is bound predominantly to one transposase monomer. We propose that a conformational change in the single-end complex, involving rotation of one half of the transposase along with binding of a second transposon end, could facilitate PEC assembly.     

Going non-viral: the Sleeping Beauty transposon system breaks on through to the clinical side

Molecular medicine has entered a high-tech age that provides curative treatments of complex genetic diseases through genetically engineered cellular medicinal products. Their clinical implementation requires the ability to stably integrate genetic information through gene transfer vectors in a safe, effective and economically viable manner. The latest generation of Sleeping Beauty (SB) transposon vectors fulfills these requirements, and may overcome limitations associated with viral gene transfer vectors and transient non-viral gene delivery approaches that are prevalent in ongoing pre-clinical and translational research. The SB system enables high-level stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, thereby representing a highly attractive gene transfer strategy for clinical use. Here we review several recent refinements of the system, including the development of optimized transposons and hyperactive SB variants, the vectorization of transposase and transposon as mRNA and DNA minicircles (MCs) to enhance performance and facilitate vector production, as well as a detailed understanding of SB’s genomic integration and biosafety features. This review also provides a perspective on the regulatory framework for clinical trials of gene delivery with SB, and illustrates the path to successful clinical implementation by using, as examples, gene therapy for age-related macular degeneration (AMD) and the engineering of chimeric antigen receptor (CAR)-modified T cells in cancer immunotherapy.     

Survey of sugar beet (Beta vulgaris L.) hAT transposons and MITE-like hATpin derivatives

Genome-wide analyses of repetitive DNA suggest a significant impact particularly of transposable elements on genome size and evolution of virtually all eukaryotic organisms. In this study, we analyzed the abundance and diversity of the hAT transposon superfamily of the sugar beet (B. vulgaris) genome, using molecular, bioinformatic and cytogenetic approaches. We identified 81 transposase-coding sequences, three of which are part of structurally intact but nonfunctional hAT transposons (BvhAT), in a B. vulgaris BAC library as well as in whole genome sequencing-derived data sets. Additionally, 116 complete and 497 truncated non-autonomous BvhAT derivatives lacking the transposase gene were in silico-detected. The 116 complete derivatives were subdivided into four BvhATpin groups each characterized by a distinct terminal inverted repeat motif. Both BvhAT and BvhATpin transposons are specific for species of the genus Beta and closely related species, showing a localization on B. vulgaris chromosomes predominantely in euchromatic regions. The lack of any BvhAT transposase function together with the high degree of degeneration observed for the BvhAT and the BvhATpin genomic fraction contrasts with the abundance and activity of autonomous and non-autonomous hAT transposons revealed in other plant species. This indicates a possible genus-specific structural and functional repression of the hAT transposon superfamily during Beta diversification and evolution.     

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.