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.     

piggyBac can bypass DNA synthesis during cut and paste transposition

DNA synthesis is considered a defining feature in the movement of transposable elements. In determining the mechanism of piggyBac transposition, an insect transposon that is being increasingly used for genome manipulation in a variety of systems including mammalian cells, we have found that DNA synthesis can be avoided during piggyBac transposition, both at the donor site following transposon excision and at the insertion site following transposon integration. We demonstrate that piggyBac transposon excision occurs through the formation of transient hairpins on the transposon ends and that piggyBac target joining occurs by the direct attack of the 3'OH transposon ends on to the target DNA. This is the same strategy for target joining used by the members of DDE superfamily of transposases and retroviral integrases. Analysis of mutant piggyBac transposases in vitro and in vivo using a piggyBac transposition system we have established in Saccharomyces cerevisiae suggests that piggyBac transposase is a member of the DDE superfamily of recombinases, an unanticipated result because of the lack of sequence similarity between piggyBac and DDE family of recombinases.     

A piggyBac transposon-based mutagenesis system for the fission yeast Schizosaccharomyces pombe

The TTAA-specific transposon piggyBac (PB), originally isolated from the cabbage looper moth, Trichoplusia ni, has been utilized as an insertional mutagenesis tool in various eukaryotic organisms. Here, we show that PB transposes in the fission yeast Schizosaccharomyces pombe and leaves almost no footprints. We developed a PB-based mutagenesis system for S. pombe by constructing a strain with a selectable transposon excision marker and an integrated transposase gene. PB transposition in this strain has low chromosomal distribution bias as shown by deep sequencing-based insertion site mapping. Using this system, we obtained loss-of-function alleles of klp5 and klp6, and a gain-of-function allele of dam1 from a screen for mutants resistant to the microtubule-destabilizing drug thiabendazole. From another screen for cdc25-22 suppressors, we obtained multiple alleles of wee1 as expected. The success of these two screens demonstrated the usefulness of this PB-mediated mutagenesis tool for fission yeast.     

Transposon-mediated mutagenesis of somatic cells in the mouse for cancer gene identification

To successfully treat cancer we will likely need a much more detailed understanding of the genes and pathways meaningfully altered in individual cancer cases. One method for achieving this goal is to derive cancers in model organisms using unbiased forward genetic screens that allow cancer gene candidate discovery. We have developed a method using a “cut-and-paste” DNA transposon system called Sleeping Beauty (SB) to perform forward genetic screens for cancer genes in mice. Although the approach is conceptually similar to the use of replication competent retroviruses for cancer gene identification, the SB system promises to allow such screens in tissues previously not amenable to forward genetic screens such as the gastrointestinal tract, brain, and liver. This article describes the strains useful for SB-based screens for cancer genes in mice and how they are deployed in an experiment.     

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth^1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect^1,2, mammalian^3-5, and human cells6 including primary human T cells^7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency^9,10.Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer¹¹. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved¹². We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene⁷. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes¹³. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model¹⁴. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized¹⁵.Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens¹⁴, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed⁷. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)¹⁵. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.     

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.     

Generation of stable Chinese hamster ovary pools yielding antibody titers of up to 7.6 g/L using the piggyBac transposon system

Chinese hamster ovary (CHO) cells remain the default production host for many biopharmaceutical drugs, particularly monoclonal antibodies (mAb). Production of gram and kilogram quantities of protein typically requires the generation of stable CHO clones. Unfortunately, this process takes several months, significantly slowing down the drug discovery and development process. Therefore, improved technologies are needed to accelerate biopharmaceutical drug discovery and final drug substance manufacturing. In this study, we describe the generation of stable CHO pools using the piggyBac transposon system. We evaluated the system using four model antibody molecules (3 mAbs and 1 bispecific Ab). Stable CHO pools were isolated in 7–12 days. Using a simple 16‐day fed‐batch process, we measured titers ranging from 2.3 to 7.6 g/L for the four model antibodies. This represented a 4‐ to 12‐fold increase relative to the controls. Additionally, we isolated stable CHO clones. We found that the stable CHO clones isolated from the piggyBac transposon pools yielded titers two to threefold higher relative to the control clones. Taken together, these results suggest that stable CHO pool and clone generation can be significantly improved by using the piggyBac transposon system. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1301–1307, 2016     

piggyBac transposon-mediated transgenesis in the apicomplexan parasite Eimeria tenella

piggyBac, a type II transposon that is useful for efficient transgenesis and insertional mutagenesis, has been used for effective and stable transfection in a wide variety of organisms. In this study we investigate the potential use of the piggyBac transposon system for forward genetics studies in the apicomplexan parasite Eimeria tenella. Using the restriction enzyme-mediated integration (REMI) method, E. tenella sporozoites were electroporated with a donor plasmid containing the enhanced yellow fluorescent protein (EYFP) gene flanked by piggyBac inverted terminal repeats (ITRs), an Asc I-linearized helper plasmid containing the transposase gene and the restriction enzyme Asc I. Subsequently, electroporated sporozoites were inoculated into chickens via the cloacal route and transfected progeny oocysts expressing EYFP were sorted by flow cytometry. A transgenic E. tenella population was selected by successive in vivo passage. Southern-blotting analysis showed that exogenous DNA containing the EYFP gene was integrated into the parasite genome at a limited number of integration sites and that the inserted part of the donor plasmid was the fragment located between the 5' and 3' ITRs as indicated by primer-specific PCR screening. Genome walking revealed that the insertion sites were TTAA-specific, which is consistent with the transposition characteristics of piggyBac.     

piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus

Transgenic insects have been artificially produced to study functions of interesting developmental genes, using insect transposons such as piggyBac. In the case of the cricket, however, transgenic animals have not yet been successfully artificially produced. In the present study, we examined whether the piggyBac transposon functions as a tool for gene delivery in embryos of Gryllus bimaculatus. We used either a piggyBac helper plasmid or a helper RNA synthesized in vitro as a transposase source. An excision assay revealed that the helper RNA was more effective in early Gryllus eggs to transpose a marker gene of eGFP than the helper plasmid containing the piggyBac transposase gene driven by the G. bimaculatus actin3/4 promoter. Further, only when the helper RNA was used, somatic transformation of the embryo with the eGFP gene was observed. These results suggest that the piggyBac system with the helper RNA may be effective for making transgenic crickets.     

A Novel Piggybac Transposon Inducible Expression System Identifies a Role for Akt Signalling in Primordial Germ Cell Migration

In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC) lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development.     

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。     

A modified piggybac transposon system mediated by exogenous mRNA to perform gene delivery in bovine mammary epithelial cells

Transposons are widely used for genetic engineering in various model organisms. Recently, piggyBac (PB) has been developed as a transposable and efficient gene transfer tool in mammalian cells. In the present study, we developed three types of PB transposon systems containing a dual plasmid system (DPS), a single plasmid system (SPS), and a DNA-mRNA combined system (DRPS) and characterized their basic properties in HEK293 cells. The basic elements of the donor plasmid included a selectable-reporter gene expression cassette, two loxP sites in the same orientation, a multiple cloning site, and two chicken β-globin insulator core elements. We further identified the function of the selectable-reporter and examined PB integration sites in the human genome. Moreover, we compared the transposition efficacy and found that SPS transposed more efficiently, as compared to DPS; integration into the host genome was determined by measuring PBase activity. Results discovered the loss of PBase activity in the DRPS, indicating that this system is much more biologically safe, as compared to DPS and SPS. Finally, we employed the DRPS to successfully perform a gene delivery into bovine mammary epithelial cells (BMECs). Taken together, the information from this study will improve the flexibility of PB transposon systems and reduce the genotoxicity of PBase in genetic engineering.     

The minimum internal and external sequence requirements for transposition of the eukaryotic transformation vector piggyBac

The piggyBac element from Trichoplusia ni is recognized as a useful vector for transgenesis of a wide variety of species. This transposable element is 2472 bp in length, and has a complex repeat configuration consisting of an internal repeat (IR), spacer, and terminal repeat (TR) at both ends, and a single ORF encoding the transposase. Excision assays performed in microinjected T. ni embryos using plasmids deleted for progressively larger portions of the piggyBac internal sequence reveal that the 5' and 3' IR, spacer, and TR configuration is sufficient for precise excision of piggyBac when transposase is provided in trans. Interplasmid transposition assays using plasmids carrying varying lengths of intervening sequence between the piggyBac termini in T. ni demonstrate that a minimum of 55 bp of intervening sequence is required for optimal transposition, while lengths less than 40 bp result in a dramatic decrease in transposition frequency. These results suggest that the piggyBac transposase may bind both termini simultaneously before cleavage can occur, and/or that the formation of a transposition complex requires DNA bending between the two termini. Based on these results we constructed a 702-bp cartridge with minimal piggyBac 5' and 3' terminal regions separated by an intervening sequence of optimal length. Interplasmid transposition assays demonstrate that the minimal terminal configuration is sufficient to mediate transposition, and also verify that simply inserting this cartridge into an existing plasmid converts that plasmid into a non-autonomous piggyBac transposon. We also constructed a minimal piggyBac vector, pXL-Bac, that contains an internal multiple cloning site sequence between the minimal terminal regions. These vectors should greatly facilitate the utilization of the piggyBac transposon in a wide range of hosts.     

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.     

Isolation of homozygous mutant mouse embryonic stem cells using a dual selection system

Obtaining random homozygous mutants in mammalian cells for forward genetic studies has always been problematic due to the diploid genome. With one mutation per cell, only one allele of an autosomal gene can be disrupted, and the resulting heterozygous mutant is unlikely to display a phenotype. In cells with a genetic background deficient for the Bloom's syndrome helicase, such heterozygous mutants segregate homozygous daughter cells at a low frequency due to an elevated rate of crossover following mitotic recombination between homologous chromosomes. We constructed DNA vectors that are selectable based on their copy number and used these to isolate these rare homozygous mutant cells independent of their phenotype. We use the piggyBac transposon to limit the initial mutagenesis to one copy per cell, and select for cells that have increased the transposon copy number to two or more. This yields homozygous mutants with two allelic mutations, but also cells that have duplicated the mutant chromosome and become aneuploid during culture. On average, 26% of the copy number gain events occur by the mitotic recombination pathway. We obtained homozygous cells from 40% of the heterozygous mutants tested. This method can provide homozygous mammalian loss-of-function mutants for forward genetic applications.     

Mobility of the piggyBac transposon in embryos of the vectors of Dengue fever (Aedes albopictus) and La Crosse encephalitis (Ae. triseriatus)

The re-emergence of arboviral diseases such as Dengue Fever and La Crosse encephalitis is primarily due to the failure of insect vector control strategies. The development of a procedure capable of producing stable germ-line transformants in the insect vectors of these diseases would bridge the gap between gene expression systems being developed to curb vector transmission and the identification of important genes and regulatory sequences and their reintroduction back into the insect genome in the form of vector control strategies. The transposable element piggyBac is capable of transposition in a variety of insect species, and could serve as a versatile insect transformation vector. Using plasmid-based excision and transposition assays, we report that this short-ITR transposon undergoes precise, transposase-dependent excision and transposition in embryos of Aedes albopictus and Aedes triseriatus, the vectors of Dengue fever and LaCrosse encephalitis, respectively. These assays allow us easily and rapidly to confirm and assess the potential utility of piggyBac as a gene transfer tool in a given species. piggyBac is an exceptionally mobile and versatile genetic transformation vector, comparable to other transposons currently in use for the transformation of insects. The mobility of the piggyBac element seen in both Ae. albopictus and Ae. triseriatus is further evidence that it can be employed as a germ-line vector in important insect disease vectors.     

A Genetic Screen Using the PiggyBac Transposon in Haploid Cells Identifies Parp1 as a Mediator of Olaparib Toxicity

Genetic perturbation screens have the potential to dissect a wide range of cellular phenotypes. Such screens have historically been difficult in diploid mammalian cells. The recent derivation of haploid embryonic stem cells provides an opportunity to cause loss of function mutants with a random mutagen in a mammalian cell with a normal genetic background. We describe an approach to genetic screens that exploits the highly active piggyBac transposon in haploid mammalian cells. As an example of haploid transposon (HTP) screening, we apply this approach to identifying determinants of cancer drug toxicity and resistance. In a screen for 6-thioguanine resistance we recovered components of the DNA mismatch repair pathway, a known requirement for toxicity. In a further screen for resistance to the clinical poly(ADP-ribose) polymerase (PARP) inhibitor olaparib we recovered multiple Parp1 mutants. Our results show that olaparib toxicity to normal cells is mediated predominantly via Parp1, and suggest that the clinical side effects of olaparib may be on target. The transposon mutant libraries are stable and can be readily reused to screen other drugs. The screening protocol described has several advantages over other methods such as RNA interference: it is rapid and low cost, and mutations can be easily reverted to establish causality.     

Progress with schistosome transgenesis

Genome sequences for Schistosoma japonicum and Schistosoma mansoni are now available. The schistosome genome encodes ~13,000 protein encoding genes for which the function of only a minority is understood. There is a valuable role for transgenesis in functional genomic investigations of these new schistosome gene sequences. In gain-of-function approaches, transgenesis can lead to integration of transgenes into the schistosome genome which can facilitate insertional mutagenesis screens. By contrast, transgene driven, vector-based RNA interference (RNAi) offers powerful loss-of-function manipulations. Our laboratory has focused on development of tools to facilitate schistosome transgenesis. We have investigated the utility of retroviruses and transposons to transduce schistosomes. Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) can transduce developmental stages of S. mansoni including eggs. We have also observed that the piggyBac transposon is transpositionally active in schistosomes. Approaches with both VSVG-MLV and piggyBac have resulted in somatic transgenesis and have lead to integration of active reporter transgenes into schistosome chromosomes. These findings provided the first reports of integration of reporter transgenes into schistosome chromosomes. Experience with these systems is reviewed herewith, along with findings with transgene mediated RNAi and germ line transgenesis, in addition to pioneering and earlier reports of gene manipulation for schistosomes.