Genetic screens using the piggyBac transposon

Transposons are an attractive system to use in genetic screens as they are molecularly tractable and the disrupted loci that give rise to the desired phenotype are easily mapped. We consider herein the characteristics of the piggyBac transposon system in complementing existing mammalian screen strategies, including the Sleeping Beauty transposon system. We also describe the design of the piggyBac resources that we have developed for both forward and reverse genetic screens, and the protocols we use in these experiments.     

piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART) for genetic screens in mice

Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.     

Postintegration stability of the silkworm piggyBac transposon

The piggyBac transposon is the most widely used vector for generating transgenic silkworms. The silkworm genome contains multiple piggyBac-like sequences that might influence the genetic stability of transgenic lines. To investigate the postintegration stability of piggyBac in silkworms, we used random insertion of the piggyBac [3 × p3 EGFP afm] vector to generate a W chromosome-linked transgenic silkworm, named W-T. Results of Southern blot and inverse PCR revealed the insertion of a single copy in the W chromosome of W-T at a standard TTAA insertion site. Investigation of 11 successive generations showed that all W-T females were EGFP positive and all males were EGFP negative; PCR revealed that the insertion site was unchanged in W-T offspring. These results suggested that endogenous piggyBac-like elements did not affect the stability of piggyBac inserted into the silkworm genome.     

Fluorescence-based phenotypic selection allows forward genetic screens in haploid human cells

The isolation of haploid cell lines has recently allowed the power of forward genetic screens to be applied to mammalian cells. The interest in applying this powerful genetic approach to a mammalian system is only tempered by the limited utility of these screens, if confined to lethal phenotypes. Here we expand the scope of these approaches beyond live/dead screens and show that selection for a cell surface phenotype via fluorescence-activated cell sorting can identify the key molecules in an intracellular pathway, in this case MHC class I antigen presentation. Non-lethal haploid genetic screens are widely applicable to identify genes involved in essentially any cellular pathway.     

Precise marker excision system using an animal‐derived piggyBac transposon in plants

Accurate and effective positive marker excision is indispensable for the introduction of desired mutations into the plant genome via gene targeting (GT) using a positive/negative counter selection system. In mammals, the moth‐derived piggyBac transposon system has been exploited successfully to eliminate a selectable marker from a GT locus without leaving a footprint. Here, we present evidence that the piggyBac transposon also functions in plant cells. To demonstrate the use of the piggyBac transposon for effective marker excision in plants, we designed a transposition assay system that allows the piggyBac transposition to be visualized as emerald luciferase (Eluc) luminescence in rice cells. The Eluc signal derived from piggyBac excision was observed in hyperactive piggyBac transposase‐expressing rice calli. Polymerase chain reaction, Southern blot analyses and sequencing revealed the efficient and precise transposition of piggyBac in these calli. Furthermore, we have demonstrated the excision of a selection marker from a reporter locus in T₀ plants without concomitant re‐integration of the transposon and at a high frequency (44.0% of excision events), even in the absence of negative selection.     

Mutagenesis by imprecise excision of the piggyBac transposon in Drosophila melanogaster

Mutagenesis by transposon-mediated imprecise excision is the most extensively used technique for mutagenesis in Drosophila. Although P-element is the most widely used transposon in Drosophila to generate deletion mutants, it is limited by the insertion coldspots in the genome where P-elements are rarely found. The piggyBac transposon was developed as an alternative mutagenic vector for mutagenesis of non-P-element targeted genes in Drosophila because the piggyBac transposon can more randomly integrate into the genome. Previous studies suggested that the piggyBac transposon always excises precisely from the insertion site without initiating a deletion or leaving behind an additional footprint. This unique characteristic of the piggyBac transposon facilitates reversible gene-transfer in several studies, such as the generation of induced pluripotent stem (iPS) cells from fibroblasts. However, it also raised a potential limitation of its utility in generating deletion mutants in Drosophila. In this study, we report multiple imprecise excisions of the piggyBac transposon at the sepiapterin reductase (SR) locus in Drosophila. Through imprecise excision of the piggyBac transposon inserted in the 5'-UTR of the SR gene, we generated a hypomorphic mutant allele of the SR gene which showed markedly decreased levels of SR expression. Our finding suggests that it is possible to generate deletion mutants by piggyBac transposon-mediated imprecise excision in Drosophila. However, it also suggests a limitation of piggyBac transposon-mediated reversible gene transfer for the generation of induced pluripotent stem (iPS) cells.     

Use of the piggyBac transposon for germ-line transformation of insects

Germ-line transformation of insects is now possible with four independent transposable element vector systems. Among these, the TTAA-insertion site specific transposon, piggyBac, discovered in Trichoplusia ni, is one of the most widely used. Transformations have been achieved in a wide variety of dipterans, lepidopterans, and a coleopteran, and for many species, piggyBac transposition was first tested by plasmid-based mobility assays in cell lines and embryos. All plasmid and genomic insertions are consistent with the duplication of a TTAA insertion site, and most germ-line integrations appear to be stable, though this is largely based on stable marker phenotypes. Of the vector systems presently in use for non-drosophilids, piggyBac is the only one not currently associated with a superfamily of transposable elements, though other elements exist that share its TTAA insertion site specificity. While functional piggyBac elements have only been isolated from T. ni, nearly identical elements have been discovered in a dipteran species, Bactrocera dorsalis, and closely related elements exist in another moth species, Spodoptera frugiperda. It appears that piggyBac has recently traversed insect orders by horizontal transmission, possibly mediated by a baculovirus or other viral system. This interspecies movement has important implications for the practical use of piggyBac to create transgenic insect strains for field release.     

piggyBac转座子及其在转基因昆虫中的应用

piggyBac是一种从粉纹夜蛾Trichoplusiani.中分离到的、具有TTAA插入位点特异性的DNA转座子。piggyBac可在昆虫基因组中准确切离,转化频率较高,并且不受宿主因子的限制,是目前转基因昆虫研究中应用最广的转座子载体。近年来的研究发现,piggyBac类转座子广泛分布于昆虫和其他生物基因组中。文章从piggyBac的结构、转座特性、在转基因昆虫中的应用以及piggyBac类转座子的分布等几个方面综述了piggyBac的研究进展。     

Evolution of the Xenopus piggyBac transposon family TxpB: domesticated and untamed strategies of transposon subfamilies

A new family, termed TxpB, of DNA transposons belonging to the piggyBac superfamily was found in 3 Xenopus species (Xenopus tropicalis, Xenopus laevis, and Xenopus borealis). Two TxpB subfamilies of Kobuta and Uribo1 were found in all the 3 species, and another subfamily termed Uribo2 was found in X. tropicalis. Molecular phylogenetic analyses of their open reading frames (ORFs) revealed that TxpB transposons have been maintained for over 100 Myr. Both the Uribo1 and the Uribo2 ORFs were present as multiple copies in each genome, and some of them were framed by terminal inverted repeat sequences. In contrast, all the Kobuta ORFs were present as a single copy in each genome and exhibited high evolutionary conservation, suggesting domestication of Kobuta genes by the host. Genomic insertion polymorphisms of the Uribo1 and Uribo2 transposons (nonautonomous type) were observed in a single species of X. tropicalis, indicating recent transposition events. Transfection experiments in cell culture revealed that an expression vector construct for the intact Uribo2 ORF caused precise excision of a nonautonomous Uribo2 element from the target vector construct but that for the Kobuta ORF did not. The present results support our viewpoint that some Uribo2 members are naturally active autonomous transposons, whereas Kobuta members may be domesticated by hosts.     

Moving forward in reverse: genetic technologies to enable genome-wide phenomic screens in Arabidopsis

Genome sequencing, in combination with various computational and empirical approaches to sequence annotation, has made possible the identification of more than 30,000 genes in Arabidopsis thaliana. Increasingly sophisticated genetic tools are being developed with the long-term goal of understanding how the coordinated activity of these genes gives rise to a complex organism. The combination of classical forward genetics with recently developed genome-wide, gene-indexed mutant collections is beginning to revolutionize the way in which gene functions are studied in plants. High-throughput screens using these mutant populations should provide a means to analyse plant gene functions--the phenome--on a genomic scale.     

Multigene expression in stable CHO cell pools generated with the piggyBac transposon system

Heterogenous populations of recombinant cells (cell pools) stably expressing 1–4 transgenes were generated from Chinese hamster overy (CHO) cells with the piggyBac (PB) transposon system. The cell pools produced different combinations of three model proteins—enhanced green fluorescent protein (EGFP), secreted alkaline phosphatase (SEAP), and a monoclonal IgG1 antibody. Each transgene was present on a separate PB donor plasmid with either the same or a different selection gene. In both cases, we obtained PB‐derived cell pools with higher recombinant protein yields than from cell pools generated by conventional gene delivery. In PB‐derived cell pools generated using a single selection agent, both protein production and the number of integrated copies of each transgene declined as the number of transfected transgenes increased. However, the total number of integrated transgenes was similar regardless of the number of different transgenes transfected. For PB‐derived cell pools generated by selection of each transgene with a different selection agent, the total number of integrated transgenes increased with the number of transfected transgenes. The results suggest that the generation of cell pools producing multiple recombinant proteins is feasible and that the method is more efficient when each individual transgene is selected with a different marker. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1308–1317, 2016     

Super-sized deletions: Improved transposon excision screens using a mus309 mutant background

Over the past two decades, a large collection of transposable elements inserted at various locations in the Drosophila melanogaster genome has been assembled. These transposons are frequently utilized in imprecise excision screens to generate deletions in genes of interest. In general, these screens involve genetic manipulations to combine a non-autonomous transposon and the appropriate transposase in individual male or female flies. DNA double-strand breaks are created via transposase action in both somatic and germline cells of these individuals and inaccurate repair events are recovered in the progeny. Because deletion-prone repair of transposon-induced double-strand breaks is rare, these screens generally require a significant investment of time and resources. We recently reported that conducting imprecise excision screens in mus309 mutant flies, which lack the Drosophila ortholog of the Bloom Syndrome helicase, results in an increase in both the number and size of deletions recovered. Here, we provide additional information for Drosophila researchers wishing to utilize this technique. In addition, we discuss how the general principle behind this technique can be applied in other contexts where double-strand breaks are being generated for the purpose of genome modification.     

piggyBac转座子AgoPLE1.1在黑腹果蝇种系转化中的应用

[目的]通过检测黑腹果蝇 DDrosophiila melanogaster中piggyBac(PB)转座子AgoPLE1.1的转化活性,明确AgoPLE1.1开发为昆虫转基因载体的潜力.[方法]构建AgoPLE1.1转座酶辅助质粒pAgoHsp和带有红色荧光标记的供体质粒pXLAgo-PUbDsRed,辅助质粒和供体...     

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.     

绵羊高效转基因通用型piggyBac转座子载体构建及功能验证

piggyBac (PB)是一种能在多种动物细胞中进行转座的DNA转座子,作为一种转基因工具已被广泛应用于各种哺乳动物转基因研究中。针对不同物种对PB转座子进行改造,是提升其通用性的必要手段。为构建基于绵羊细胞进行转基因操作的通用型PB转座子载体,本研究对PB转座酶(PBase)基因进行绵羊密码子偏好性优化并将其克隆到pBNW-TP1载体中,成功构建了PB转座子载体pBNW-TP2。将pBNW-TP2转染到绵羊成纤维细胞和乳腺上皮细胞中,利用G418筛选获取稳定转染细胞株;利用Tail-PCR检测稳定转染细胞株的PB转座位点,对细胞阳性克隆进行亚甲蓝染色;利用非配对t检验确认其转座效率。结果表明,pBNW-TP2成功介导了绵羊成纤维细胞和乳腺上皮细胞转基因阳性细胞株的生产;PB转座位点检测表明pBNW-TP2能特异性整合到绵羊基因组TTAA位点,其整合位点倾向于功能基因间;亚甲蓝染色统计分析结果提示pBNW-TP2介导的转基因效率显著提升。本研究成功构建了绵羊通用型PB转座子载体pBNW-TP2,并在绵羊体细胞中对其特性进行验证和分析,为PB转座子在绵羊体细胞中开展转基因相关研究提供了科学依据。     

Two forward genetic screens for vein density mutants in sorghum converge on a cytochrome P450 gene in the brassinosteroid pathway

The specification of vascular patterning in plants has interested plant biologists for many years. In the last decade a new context has emerged for this interest. Specifically, recent proposals to engineer C₄ traits into C₃ plants such as rice require an understanding of how the distinctive venation pattern in the leaves of C₄ plants is determined. High vein density with Kranz anatomy, whereby photosynthetic cells are arranged in encircling layers around vascular bundles, is one of the major traits that differentiate C₄ species from C₃ species. To identify genetic factors that specify C₄ leaf anatomy, we generated ethyl methanesulfonate‐ and γ‐ray‐mutagenized populations of the C₄ species sorghum (Sorghum bicolor), and screened for lines with reduced vein density. Two mutations were identified that conferred low vein density. Both mutations segregated in backcrossed F₂ populations as homozygous recessive alleles. Bulk segregant analysis using next‐generation sequencing revealed that, in both cases, the mutant phenotype was associated with mutations in the CYP90D2 gene, which encodes an enzyme in the brassinosteroid biosynthesis pathway. Lack of complementation in allelism tests confirmed this result. These data indicate that the brassinosteroid pathway promotes high vein density in the sorghum leaf, and suggest that differences between C₄ and C₃ leaf anatomy may arise in part through differential activity of this pathway in the two leaf types.