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...     

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.     

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.     

Comparison of transformation efficiency of piggyBac transposon among three different silkworm Bombyx mori Strains

The transformation rate of three different strains of silkworm Bombyx mori was comparedafter the introduction of enhanced green fluorescence protein (EGFP)-encoding genes into the silkwormeggs by microinjection of a mixture of piggyBac vector and helper plasmid containing a transposase-encodingsequence.Although there were no significant differences among the three strains in the percentages offertile moths in microinjected eggs (P=0.1258),the percentages of G_0 transformed moths in fertile mothsand injected eggs were both significantly different (P=0.01368 and P=0.02398, respectively).Thetransformation rate of the Nistari strain (Indian strain) was significantly higher than that of the other twostrains,Golden-yellow-cocoon (Vietnamese strain) and Jiaqiu (Chinese strain),which had similar rate. Theseresults indicate that the transformation efficiency of the piggyBac-based system might vary with silkwormstrains with different genetic backgrounds.The presence of endogenous piggyBac-like elements might bean important factor influencing the transformation efficiency of introduced piggyBac-derived vectors,andthe diverse amount and activation in different silkworm strains might account for the significant differences.     

Germline transformation of the spotted wing drosophilid, Drosophila suzukii, with a piggyBac transposon vector

Drosophila suzukii is a pest of small fruits in many parts of the world, whose management is limited to cultural practices and the use of insecticides. Here we describe a method to genetically manipulate this species in the first step to create female lethality strains useful for the sterile insect technique method of population suppression. This was achieved by the germ-line transformation of D. suzukii with a piggyBac transposon vector having a female-specific lethality effector construct. This can be used in a tetracycline-suppressible conditional gene expression system, when crossed to a suitable tet-transactivator strain. Transformation occurred efficiently, at a frequency of 16 % per fertile G0 embryo injected with vector and helper transposase plasmids. The vector was marked for transformant selection with the polyubiquitin-regulated EGFP fluorescent protein, and contains the attP landing site and heterospecific lox recombination sites for post-integration modification of the transgene vector. The 3xP3-AmCyan fluorescent protein marker was inserted within the lox sites to follow a possible recombinase-mediated cassette exchange, that would allow subsequent improvement of the transgenic strain by immobilization of the vector and introduction of new marker cassettes.     

Stable transformation of a Mamestra brassicae (lepidoptera) cell line with the lepidopteran-derived transposon piggyBac

Cabbage moth cells were transfected with the vector pBac[3xP3-EGFPafm] and helper phsp-pBac. Seventeen percent of the transfected cells showed stable EGFP-expression. This indicates successful and stable transformation of M. brassicae cells with a piggyBac-derived vector. Genomic integration of Bac[3xP3-EGFPafm] in stably transformed cells was confirmed by Southern blots and inverse PCR. Since the integrations are stable, and transfection with pBac[3xP3-EGFPafm] alone did not yield in transformations, no cross-reacting transposase activity seems present in M. brassicae cells. Moreover, Southern blotting with a probe for piggyBac transposase indicated the absence of piggyBac-related elements in the genome of Mamestra brassicae. Due to the tissue specificity of the 3xP3-EGFP marker for eye and nervous tissues, it is intriguing that 3xP3-EGFP can successfully be used to identify stably transformed M. brassicae cells of cell line IZD-MB0503, which is hemocyte-derived. Sequence analysis of the insertion sites showed that piggyBac inverted repeats were adjacent to TTAA sequences on both termini in all the clones. The present results are particularly important as they suggest that piggyBac can be used for transgenesis of cabbage moth cells.     

Generation of an inducible and optimized piggyBac transposon system

Genomic studies in the mouse have been slowed by the lack of transposon-mediated mutagenesis. However, since the resurrection of Sleeping Beauty (SB), the possibility of performing forward genetics in mice has been reinforced. Recently, piggyBac (PB), a functional transposon from insects, was also described to work in mammals. As the activity of PB is higher than that of SB11 and SB12, two hyperactive SB transposases, we have characterized and improved the PB system in mouse ES cells. We have generated a mouse codon-optimized version of the PB transposase coding sequence (CDS) which provides transposition levels greater than the original. We have also found that the promoter sequence predicted in the 5′-terminal repeat of the PB transposon is active in the mammalian context. Finally, we have engineered inducible versions of the optimized piggyBac transposase fused with ERT2. One of them, when induced, provides higher levels of transposition than the native piggyBac CDS, whereas in the absence of induction its activity is indistinguishable from background. We expect that these tools, adaptable to perform mouse-germline mutagenesis, will facilitate the identification of genes involved in pathological and physiological processes, such as cancer or ES cell differentiation.     

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.     

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.     

Toward forward genetic screens in malaria-causing parasites using the piggyBac transposon

The ability to analyze gene function in malaria-causing Plasmodium parasites has received a boost with a recent paper in BMC Genomics that describes a genome-wide mutagenesis system in the rodent malaria species Plasmodium berghei using the transposon piggyBac. This advance holds promise for identifying and validating new targets for intervention against malaria. But further improvements are still needed for the full power of genome-wide molecular genetic screens to be utilized in this organism.     

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.     

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。     

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     

High efficiency germ-line transformation of mosquitoes

The ability to manipulate the mosquito genome through germ-line transformation provides us with a powerful tool for investigating gene structure and function. It is also a valuable method for the development of novel approaches to combating the spread of mosquito-vectored diseases. To date, germ-line transformation has been demonstrated in several mosquito species. Transgenes are introduced into pre-blastocyst mosquito embryos using microinjection techniques that take a few hours, and progeny are screened for the presence of a marker gene. The microinjection protocol presented here can be applied to most mosquitoes and contains several improvements over other published methods that increase the survival of injected embryos and, therefore, the number of transformants. Transgenic lines can be established in approximately 1 month using this technique.     

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。     

Bioreactor scale up and protein product quality characterization of piggyBac transposon derived CHO pools

Chinese hamster ovary (CHO) cells remain the most popular host for the production of biopharmaceutical drugs, particularly monoclonal antibodies (mAbs), bispecific antibodies, and Fc‐fusion proteins. Creating and characterizing the stable CHO clonally‐derived cell lines (CDCLs) needed to manufacture these therapeutic proteins is a lengthy and laborious process. Therefore, CHO pools have increasingly been used to rapidly produce protein to support and enable preclinical drug development. We recently described the generation of CHO pools yielding mAb titers as high as 7.6 g/L in a 16 day bioprocess using piggyBac transposon‐mediated gene integration. In this study, we wanted to understand why the piggyBac pool titers were significantly higher (2–10 fold) than the control CHO pools. Higher titers were the result of a combination of increased average gene copy number, significantly higher messenger RNA levels and the homogeneity (i.e. less diverse population distribution) of the piggyBac pools, relative to the control pools. In order to validate the use of piggyBac pools to support preclinical drug development, we then performed an in‐depth product quality analysis of purified protein. The product quality of protein obtained from the piggyBac pools was very similar to the product quality profile of protein obtained from the control pools. Finally, we demonstrated the scalability of these pools from shake flasks to 36L bioreactors. Overall, these results suggest that gram quantities of therapeutic protein can be rapidly obtained from piggyBac CHO pools without significantly changing product quality attributes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:534–540, 2017     

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.     

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.