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。     

<Emphasis Type="Italic">Tβ4</Emphasis>-overexpression based on the piggyBac transposon system in cashmere goats alters hair fiber characteristics

Increasing cashmere yield is one of the vital aims of cashmere goats breeding. Compared to traditional breeding methods, transgenic technology is more efficient and the piggyBac (PB) transposon system has been widely applied to generate transgenic animals. For the present study, donor fibroblasts were stably transfected via a PB donor vector containing the coding sequence of cashmere goat thymosin beta-4 (Tβ4) and driven by a hair follicle-specific promoter, the keratin-associated protein 6.1 (KAP6.1) promoter. To obtain genetically modified cells as nuclear donors, we co-transfected donor vectors into fetal fibroblasts of cashmere goats. Five transgenic cashmere goats were generated following somatic cell nuclear transfer (SCNT). Via determination of the copy numbers and integration sites, the Tβ4 gene was successfully inserted into the goat genome. Histological examination of skin tissue revealed that Tβ4-overexpressing, transgenic goats had a higher secondary to primary hair follicle (S/P) ratio compared to wild type goats. This indicates that Tβ4-overexpressing goats possess increased numbers of secondary hair follicles (SHF). Our results indicate that Tβ4-overexpression in cashmere goats could be a feasible strategy to increase cashmere yield.     

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.     

Application of the Sleeping Beauty system in Saanen goat fibroblast cells for establishing persistent transgene expression

The Sleeping Beauty (SB) transposon system is a promising new method for establishing persistent transgene expression in vivo. We applied the SB system for enhancing transgenesis in Saanen dairy goat fibroblast cells. We constructed a pKT2/CMV-EGFP-IRES-PURO vector and investigated the influence of transposon and transposase vector ratios on transfection efficiency in the Saanen goat fibroblast cells. To enhance the SB system performance, we developed a new transfection technique (double-transfection method) for the SB system. The cultured cells were transfected with transposase and transposon vectors successively, with a 42-h interval. Consequently, the transposase and DNA donor (transposon vector) can interact, both at the highest level. Compared with the traditional transfection method, this new double-transfection method approximately doubled integration efficiency.     

一种通用型piggyBac转座子诱导细胞永生化载体的构建及其基本功能验证

为了构建一种高效、通用的细胞永生化载体pTP-hTERT,通过人工合成、PCR、酶切连接等方法,对传统piggyBac(PB)转座子系统进行改造。改造后的载体含有转座必需元件、PB转座酶(PBase)表达框、共表达筛选元件和人端粒逆转录酶(hTERT)基因表达框。其中筛选元件中绿色荧光蛋白(EGFP)基因和嘌呤霉素抗性(Puror)基因以猪捷申病毒2A自我剪切肽相连,以实现共表达。为验证载体元件功能,使用该载体转染HEK 293细胞,并对筛选出的阳性细胞进行RT-PCR、Western blotting(WB)、热不对称PCR(Tail-PCR)和细胞克隆亚甲蓝染色与统计分析。载体测序鉴定与细胞培养结果表明,通用型永生化载体pTP-hTERT构建成功,转染HEK293细胞后能筛选出抗嘌呤霉素细胞单克隆;WB结果显示P2A可高效切割EGFP和Puror融合蛋白,证明筛选标记基因功能正常;Tail-PCR结果表明该载体以转座整合插入宿主基因组;亚甲蓝染色统计结果显示由pTP-hTERT引发的细胞阳性克隆数与对照组相比显著提高(P0.01)。PB转座子永生化载体pTP-hTERT的构建为永生化细胞系的建立提供了工具,同时也为其他真核载体的构建和改造提供了参考。     

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.     

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。     

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Drosophila 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,辅助质粒和供体质粒以170 ng/μL∶400 ng/μL, 90 ng/μL∶200 ng/μL和90 ng/μL∶100 ng/μL 3种不同的比例混合后分别注射新鲜的W¹¹¹⁸ 黑腹果蝇胚胎,筛选注射后代中的转基因黑腹果蝇个体;利用Southern杂交验证转基因黑腹果蝇中AgoPLE1.1转座子的插入拷贝数;利用染色体步移技术克隆AgoPLE1.1插入位点旁侧序列,明确AgoPLE1.1转座子的转座特征。【结果】AgoPLE1.1转座子在黑腹果蝇中具有转化活性,转基因频率为1.32%~1.94%。Southern杂交结果显示,AgoPLE1.1转座子在黑腹果蝇中至少有6个插入位点。染色体步移法克隆了其中4个位点,分别位于黑腹果蝇的3R, 3L, 2L和X染色体,并且AgoPLE1.1转座子在黑腹果蝇染色体中的整合带有供体质粒的骨架。【结论】PB转座子AgoPLE1.1仅可以在黑腹果蝇中以较低的频率进行非精确的剪切和转座,不具有开发为新型昆虫转基因载体的潜力。     

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。     

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.     

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.     

Efficient production of transgenic chickens based on piggyBac

Transgenic techniques in chickens have been developed much more slowly than in mammals due to chickens’ unique reproduction mechanism. Retroviral methods have been the most successful. piggyBac (PB) is a transposon that has a 13 bp perfect terminal invert repeat sequence. PB can be inserted into TTAA sites and can also be precisely excised in mammals. Therefore, we have selected PB as a candidate to establish a new method to produce transgenic chickens. We constructed three donor vectors (ZGl-neo, ZGm-neo and ZGs-neo) expressing a GFP marker-gene and a neomycin resistant gene based on PB. We co-transfected each donor vector with a helper vector (CAG-PBase). We found that ZGl-neo was the most efficient PB vector. This vector could insert into TTAA sites in DF-1 cells. PB vectors were microinjected into sub-germinal cavity of newly laid eggs, and electroporation was then performed with a 20-V pulse for 5 cycles of 50 ms on and 100 ms off. GFP was expressed in different tissues of the embryos, including the gonads. Twenty-two chickens hatched after microinjection with compounds ZGl-neo and CAG-PBase (3:1). When we screened the blood DNA, 73 % (16/22) of the individuals were positive. Thirteen of the chickens grew to adulthood, 11 of which were males. 40 % (4/10) of the individuals were semen positive, and their copy numbers ranged from 0.05 to 0.21 (0.11, 0.21, 0.05, 0.06). No G1 offspring containing the integrated transposon were produced. We conclude that the PB transposon system is a novel useful tool for the efficient production of transgenic chickens.     

The PiggyBac transposon enhances the frequency of CHO stable cell line generation and yields recombinant lines with superior productivity and stability

Generating stable, high-producing mammalian cell lines is a major bottleneck in the manufacture of recombinant therapeutic proteins. Conventional gene transfer methods for cell line generation rely on random plasmid integration, resulting in unpredictable and highly variable levels of transgene expression. As a consequence, a large number of stably transfected cells must be analyzed to recover a few high-producing clones. Here we present an alternative gene transfer method for cell line generation based on transgene integration mediated by the piggyBac (PB) transposon. Recombinant Chinese hamster ovary (CHO) cell lines expressing a tumor necrosis factor receptor:Fc fusion protein were generated either by PB transposition or by conventional transfection. Polyclonal populations and isolated clonal cell lines were characterized for the level and stability of transgene expression for up to 3 months in serum-free suspension culture. Pools of transposed cells produced up to fourfold more recombinant protein than did the pools generated by standard transfection. For clonal cell lines, the frequency of high-producers was greater following transposition as compared to standard transfection, and these clones had a higher volumetric productivity and a greater number of integrated transgenes than did those generated by standard transfection. In general, the volumetric productivity of the cell pools and individual cell lines generated by transposition was stable for up to 3 months in the absence of selection. Our results indicate that the PB transposon supports the generation of cell lines with high and stable transgene expression at an elevated frequency relative to conventional transfection. Thus, PB-mediated gene delivery is expected to reduce the extent of recombinant cell line screening.     

Development of hybrid baculovirus vectors for artificial MicroRNA delivery and prolonged gene suppression

MicroRNA (miRNA) plays essential roles in regulating gene expression, but miRNA delivery remains a hurdle, thus entailing a vector system for efficient transfer. Baculovirus emerges as a promising gene delivery vector but its inherent transient expression restricts its applications in some scenarios. Therefore, this study primarily aimed to develop baculovirus as a miRNA expression vector for prolonged gene suppression. We constructed recombinant baculoviruses carrying artificial egfp-targeting miRNA sequences within the miR155 backbone, which after expression by the cytomegalovirus promoter could knockdown the enhanced green fluorescent protein (EGFP) expression in a sequence- and dose-dependent manner. By swapping the mature miRNA sequences, the baculovirus miRNA shuttle effectively repressed the overexpression of endogenous TNF-α in arthritic synoviocytes without inducing apoptosis. To prolong the baculovirus-mediated expression, we further developed a hybrid baculovirus vector that exploited the Sleeping Beauty (SB) transposon for gene integration and sustained miRNA expression. The hybrid baculovirus vector that combined the miR155 scaffold and SB transposon effectively repressed the transgene expression for a prolonged period of time, hence diversifying the applications of baculovirus to indications necessitating prolonged gene regulation such as arthritis.     

The piggyBac transposon is an integrating non-viral gene transfer vector that enhances the efficiency of GDEPT

Gene-directed enzyme prodrug therapy (GDEPT) is a strategy developed to selectively target cancer cells. However, the clinical benefit is limited due to its poor gene transfer efficiency. To overcome this obstacle, we took advantage of piggyBac (PB) transposon, a natural non-viral gene vector that can induce stable chromosomal integration and persistent gene expression in vertebrate cells, including human cells. To determine whether the vector can also mediate stable gene expression in ovarian cancer cells, we constructed a PB transposon system that simultaneously expresses the Herpes simplex virus thymidine kinase (HSV-tk) gene and the monomeric red fluorescent protein (mRFP1) reporter gene. The recombinant plasmid, pPB/TK, was transfected into ovarian adenocarcinoma cells SKOV3 with FuGENE HD reagent, and the efficiency was given by the percentage of mRFP1-positive cells detected by flow cytometry and confocal microscopy. The specific expression of HSV-tk in transfected cells was confirmed by RT-PCR and western blotting. The sensitivity of transfected cells to pro-drug ganciclovir (GCV) was determined by methylthiazoletetrazolium (MTT) assay. A total of 56.4 ± 8.4% cells transfected with pPB/TK were mRFP1 positive, compared to no measurable mRFP1 expression in pORF-HSVtk-transfected cells. The expression level of HSV-tk in pPB/TK-transfected cells was ∼10 times higher than in pORF-HSVtk-transfected cells. The results show that pPB/TK transfection increases the sensitivity of cells to GCV in a dose-dependent manner. Our data indicate that the PB transposon system could enhance the anti-tumor efficiency of GDEPT in ovarian cancer.     

piggyBac转座系统在哺乳动物及其细胞中的研究进展

piggyBac（PB）转座系统来源于昆虫鳞翅目,属于真核生物的第二类转座系统,主要采取＂剪切粘帖＂机制发生转座。PB系统转座效率高,宿主范围广,广泛应用于昆虫等低等生物的基因转移及突变筛选。近年来,研究发现PB系统在哺乳动物及其细胞中也具有高效的转座活性,已在动物基因组功能研究、基因转移及诱导多能干细胞等领域得到了广泛应用。本文就PB系统近年来在哺乳动物及其细胞中的研究进展、应用前景及存在问题进行了综述。     

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.     

Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo

A major limitation of adenovirus-mediated gene therapy for inherited diseases is the instability of transgene expression in vivo, which originates at least in part from the loss of the linear, extrachromosomal vector genomes. Herein we describe the production of a gene-deleted adenovirus-transposon vector that stably maintains virus-encoded transgenes in vivo through integration into host cell chromosomes. This system utilizes a donor transposon vector that undergoes Flp-mediated recombination and excision of its therapeutic payload in the presence of the Flp and Sleeping Beauty recombinases. Systemic in vivo delivery of this system resulted in efficient generation of transposon circles and stable transposase-mediated integration in mouse liver. Somatic integration was sufficient to maintain therapeutic levels of human coagulation Factor IX for more than six months in mice undergoing extensive liver proliferation. These vectors combine the versatility of adenoviral vectors with the integration capabilities of a eukaryotic DNA transposon and should prove useful in the treatment of genetic diseases.     

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