Improvement in Tol2 transposon for efficient large-cargo capacity transgene applications in cultured cells and zebrafish(Danio rerio)

Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes.However,their activity markedly diminishes with payloads exceeding 11 kb.Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs,improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics,metabolic engineering,and transgenic animal production.In this study,we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer(QBI SP163,ST)and enhanced the nuclear targeting ability using the nuclear localization protein H2B(SHT).The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures(H1299),comparable to the well-established super PiggyBac system.Furthermore,mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads(8 kb,14 kb,and 24 kb)into zebrafish(Danio rerio).This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications.

Lung-Targeted Transgene Expression of Nanocomplexed Ad5 Enhances Immune Response in the Presence of Preexisting Immunity

Recombinant adenovirus serotype 5(Ad5)vector has been widely applied in vaccine development targeting infectious diseases,such as Ebola virus disease and coronavirus disease 2019(COVID-19).However,the high prevalence of preexisting anti-vector immunity compromises the immunogenicity of Ad5-based vaccines.Thus,there is a substantial unmet need to minimize preexisting immunity while improving the insert-induced immunity of Ad5 vectors.Herein,we address this need by utilizing biocompatible nanoparticles to modulate Ad5–host interactions.We show that positively charged human serum albumin nanoparticles((+)HSAnp),which are capable of forming a complex with Ad5,significantly increase the transgene expression of Ad5 in both coxsackievirus–adenovirus receptor-positive and-negative cells.Furthermore,in charge-and dose-dependent manners,Ad5/(+)HSAnp complexes achieve robust(up to227-fold higher)and long-term(up to 60 days)transgene expression in the lungs of mice following intranasal instillation.Importantly,in the presence of preexisting anti-Ad5 immunity,complexed Ad5-based Ebola and COVID-19 vaccines significantly enhance antigen-specific humoral response and mucosal immunity.These findings suggest that viral aggregation and charge modification could be leveraged to engineer enhanced viral vectors for vaccines and gene therapies.

See the color,see the seed:GmW1 as a visual reporter for transgene and genome editing in soybean

A fast and efficient recognition method of transgenic lines will greatly improve detection efficiency and reduce cost.In this study,we successfully identified the transgenic soybean plants by the color.We isolated a GmW1 gene encoding a flavonoid 3'5'-hydroxylase from a soybean cultivar ZH42(purple flower).We found that purple flowers occurred in the overexpression lines in the Jack and Williams 82 backgrounds(white flower).All plants with purple flowers were positive,and this trait seems stably inherited in the offspring.We have also obtained the editing plants,which were classified into three types according to the different flower colors appeared.We analyzed the phenotype and the homozygous types of the T_1mutants.We also found that a correspondence between flower color and stem color.This study provides a visible color reporter on soybean transformation.It can be quickly and early to identify the transgenic soybean plants by stem color of seedlings,which substantially reduces the amount of labor and cost.

Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR(TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35 S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice.

Expression profiling of transgenes(Cry1Ac and Cry2A) in cotton genotypes under different genetic backgrounds

Transgenic cotton carrying the CrylAc gene has revolutionized insect pest control since its adoption,although the development of resistance in insect pests has reduced its efficacy.After 10 years of cultivating Bacillus thuringiensis(Bt)cotton with a single Cry1 Ac gene,growers are on the verge of adopting Bt cotton that carries the double gene(Cry1 Ac+Cry2 A)due to its better effectiveness against insect pests.Thus,the current study was designed to evaluate the role of each gene in the effectiveness of Bt cotton carrying the double gene.The expression levels of the Cry1 Ac and Cry2 A genes were evaluated in the leaves of 10 genotypes(2 parents and 8 Fhybrids)at 30 days after sowing(DAS),while samples of leaves,bolls and flowers were taken from the upper and lower canopies at 70 and 110 DAS.The Fhybrids were developed through reciprocal crosses between two Bt(CKC-1,CKC-2)and two non-Bt(MNH-786,FH-942)parents.The differential expression of transgenes was evaluated through Enzyme Linked Immuno-Sorbent Assay(ELISA).The results showed that the MNH786 xCKC-1 hybrid had the highest concentrations of Cry1 Ac gene at30 DAS(3.08μg g^(-1))and 110 DAS(1.01μg g^(-1))in leaves.In contrast,the CKC-2 xMNH-786 hybrid showed the lowest concentrations of Cry1 Ac gene at 30 DAS(2.30μg g^(-1))and 110 DAS(0.86μg g^(-1)).The Fhybrid FH-942×CKC-2 showed the highest concentrations of Cry2 A gene at 30 DAS(8.39μg g^(-1))and 110 DAS(7.74μg g^(-1))in leaves,while the CKC-1 xMNH-786 hybrid expressed the lowest concentrations of Cry2 A gene at 30 DAS(7.10μg g^(-1))and 110 DAS(8.31μg g^(-1)).A comparison between the two stages of plant growth showed that leaves had the highest concentrations at 30 DAS,whereas the lowest concentrations were observed at 110 DAS for both genes in leaves.When the expression pattern was compared between various plant parts in genotype CKC-2,it was found that leaves had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))at 70 DAS,followed by bolls(Cry1 Ac(1.66μg g^(-1))and Cry2 A(8.15μg g^(-1)))and flowers(Cry1 Ac(1.07μg g^(-1))and Cry2 A(7.99μg g^(-1))).The genotype CKC-2 had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))in the upper canopy but less accumulation(2.66μg g^(-1)of Cry1 Ac,8.09μg g^(-1)of Cry2 A)in the lower canopy at 70 DAS.Similarly,at 110 DAS,the expression levels of Cry1 Ac and Cry2 A in upper and lower canopy leaves were 1.52 and 7.92μg 9,and 0.99 and 7.54μg 9,respectively.Hence,the current study demonstrates that different genotypes showed variable expression for both of the Cry1 Ac and Cry2 A genes during plant growth due to different genetic backgrounds.The Cry2 A gene had three-fold higher expression than Cry1 Ac with significant differences in expression in different plant parts.The findings of this study will be helpful for breeding insect-resistant double-gene genotypes with better gene expression levels of Cry1 Ac and Cry2 A for sustainable cotton production worldwide.

Transgene Stacking in Cotton Improvement

To date,more and more transgenic varieties of upland cotton(Gossypium hirsutum L.) generated with transgenes,which derived from varies of alien species,are playing important role in agricultural production.Stacking of multi-transgenes has a potential for combining all the merits of

Identification of transgene insertions in two genetically modified soybeans using high throughput next generation sequencing

Genetically modified(GM) organisms are widely adopted. However, their safety assessments and control are still of special concern to the public. Identifying and localizing transgene insertion is an essentially prerequisite step. In this study, 2 independent transgene soybean lines were selected(LB4-AtDCGS-1-20-5-2 and CGS-ZG11) as typical cases. Both lines contained expression cassette of At-DCGS that encoding a feedback-insensitive cystathionine gamma-synthase to produce higher level methionine(Met). LB4-AtDCGS-1-20-5-2 was whole genome sequenced with one paired-end 500 bp library and two mate-paired 1 kb and 2 kb libraries using Illumina HiSeq sequencing platform. CGS-ZG11 was sequenced with only one paired-end 500 bp library. Both genomes were assembled,and 2 scaffold sequences(1 for each line) were screened out by aligning with transgene.Then the transgene insertion and its flanking regions in soybean genome were further identified and confirmed by PCR cloning and Sanger sequencing. Results showed that these 2 transgene lines had single copy of inserted transgene. Their transgene insertion contents were identified, which facilitates further safety assessment. These results indicated that genome assembly using high throughput sequencing is a powerful tool for identifying transgene insertions, even with limited knowledge.

01086 Transgene的抗肿瘤药需要基金

法国生物技术公司Transgene有许多有希望的抗肿瘤计划要开始Ⅲ期试验，但它需要找到一个合作者使这些计划进行下去。公司的产品从3个主要方面对抗肿瘤：即癌疫苗，免疫系统刺激剂和应用自杀基因导向性化疗。公司已经与一些可能的合作者开始了最初的讨论。

Minor modifications in obtainable Arabidopsis floral dip method enhances transformation efficiency and production of homozygous transgenic lines harboring a single copy of transgene

Many researchers have developed various methods for in-planta or floral dip transformation of Arabidopsis thaliana, one of the simple protocol and widely used to produce transgenic Arabidopsis. As the efficiency and ease of getting a transformant is very much time consuming effort and less number of the transformants people get, we have developed a little modified transformation protocol to avoid the disparities. Four types of inoculums (inoculum1, inoculum2, inoculum3 and inoculum4) were used to check the transformation efficiency out of which Inoculum3 showed the highest rate of transformation among the four types. 0.07% Twin-20 also acts in same manner as silwet L-77 to increase the rate of transformation efficiency and glucose instead of sucrose can be used in inoculum to transform Arabidopsis. After vacuum infiltration keeping the Agrobacterium infected plants for 7-8 hrs horizontally in low light at 280C temperature condition, considered best to get an increased number of transformed seeds. Modified protocol produced ~12-14% increase in transformants. Selection pots (kanamycin supplemented soil filled pots) in place of selection plates (Kanamycin supplemented Murashige and Skoog agar plates) proved beneficial as no MS medium and no aseptic condition is required for selection of transformed plants. This increase in transformation efficiency consequently increased the percentage of homozygous and single copied stable transgenic lines.

Indication of the Expression of Transgene in Rice Plant Based on Hyperspectral Remote Sensing Technique Ⅱ——Growth Monitoring of Samples in the Contrast Experiment

Since the complication of monitoring and evaluating the problems about the transgenic expression and its impacts on the receptor in the transgenic crop breeding and other relevant evaluated works,the authors in the present work tried to assess the differences of spectral parameters of the transgenic rice in contrast with its parent group quantitatively and qualitatively,fulfilling the growth monitoring of the transgenic samples.The spectral parameters(spectral morphological characteristics and indices) chosen are highly related to internal or external stresses to the receipts,and thus could be applied as indicators of biophysical or biochemical processes changes of plant.By ASD portable field spectroradiometer with high-density probe,fine foliar spectra of 8 groups were obtained.By analyzing spectral angle and continuum removal,the spectral morphological differences and their locations of sample spectra were found which could be as auxiliary priori knowledge for quantitative analysis.By investigating spectral indices of the samples,the quantitative differences of spectra were revealed about foliar chlorophyll a+b and carotenoid content.In this study both the spectral differences between transgenic and parent groups and among transgenic groups were investigated.The results show that hyperspectral technique is promising and a helpful auxiliary tool in the study of monitoring the transgenic crop and other relevant researches.By this technique,quantitative and qualitative results of sample spectra could be provided as prior knowledge,as certain orientation,for laboratory professional advanced transgenic breeding study.

Transgene Flow from Glufosinate-Resistant Rice to Improved and Weedy Rice in China

The development of transgenic rice with novel traits in China can increase rice productivity, but transgene flow to improved or weedy rice has become a major concern. We aimed to evaluate the potential maximum frequencies of transgene flow from glufosinate-resistant rice to improved rice cultivars and weedy rice. Treatments were arranged in randomized complete blocks with three replicates. Experiments were conducted between 2009 and 2010 at the Center for Environmental Safety Supervision and Inspection for Genetically Modified Plants, China National Rice Research Institute, Hangzhou, China. Glufosinate-resistant japonica rice 99-1 was the pollen donor. The pollen recipients were two inbred japonica rice （Chunjiang 016 and Xiushui 09）, two inbred indica rice （Zhongzu 14 and Zhongzao 22）, two indica hybrid rice （Zhongzheyou 1 and Guodao 1）, and one weedy indica rice （Taizhou weedy rice）. The offspring of recipients were planted in the field and sprayed with a commercial dose of glufosinate. Leaf tissues of survivors were analyzed by polymerase chain reaction to detect the presence of the transgene. The frequency of gene flow ranged from 0 to 0.488%. In 2009, the order of gene flow frequency was as follows： weedy rice 〉 Chunjiang 016 〉 Xiushui 09 and Zhongzu 14 〉 Guodao 1, Zhongzheyou 1 and Zhongzao 22. Gene flow frequencies were generally higher in 2009 than in 2010, but did not differ significantly among rice materials. Gene flow frequency was the highest in weedy rice followed by the inbred japonica rice. The risk of gene flow differed significantly between years and year-to-year variance could mask risk differences among pollen recipients. Gene flow was generally lesser in taller pollen recipients than in shorter ones, but plant height only accounted for about 30% of variation in gene flow. When flowering synchrony was maximized, as in this study, low frequencies of gene flow occurred from herbicide-resistant japonica rice to other cultivars and weedy rice. Averaged across years, the risk of gene flow to weedy rice was higher than that of improved rice and hybrids. Greater resources must be dedicated to the management of remnant weedy rice in fields planted with herbicide-resistant rice, and to prevent the evolution of resistant weedy rice populations.

INFLUENCE OF CHEMOTHERAPEUTANTS AND CYTOKINES ONGROWTH AND TRANSGENE EXPRESSION OF BONE MARROWCELLS FROM MT/P210^(bcr-ab1) TRANSGENIC MICE

Objective: To investigate the influcnce ofchemotherapeutic agents and cytokines on growth ofbone marrow cells from MT/p210 her-ab1 transgenic mice.Methods: The bone marrow cells of transgenic chronicmyelogenous leukemia (CML) model mice carryingmetallothionein (MT) promoter/enhancer, her-abl (p210)cDNA and SV40 splicinglpoly (A) signal sequences werecultured in liquid and soft agar with hydroxyurea (Hu),5-nuorouracil (5-Fu), mouse stem cell factor (mSCF)and mouse interleukin-3 (mIL-3) independently orcollectively. The cells and colonies were counted. Thelevels of transgene expression were detected by reversetranscriptase-polymerase chain reaction (RT-PCR).Results: The cell proliferation, colony formation andtransgene expression of the bone marrow cells werestimulated with mSCF and mIL-3, but there was littlegrowth without any growth factors, or when mSCF,mIL-3 and Hu or 5-Fu were added. Conclusion: Thecombined utilization of chemotherapeutants andcytokines is a potentially effective strategy of clinicaltreatment for CML.

Pronuclear microinjection is not suitable for RNA polymerase III promoter driven constitutive RNAi transgenesis in mice for XY male-to-female sex reversal by <i>Sry</i>gene knockdown

Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard methods of shRNA transgenic (Tg) mice production have not been established. Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coitus (dpc) triggers the differentiation of Sertoli cells from granulosa cells. Then high efficiency of Sry gene silencing in Tg mice should induce XY male-to-female sex reversal. An shRNA Tg mouse targeting Sry gene was attempted to be generated by pronuclear microinjection. A low rate (Tg pups/all pups born after microinjection = 2/154 to 7/178) of Tg pups was observed. These Tg mice showed no XY male-to-female sex reversal. The results suggest that exogenous expression of small RNA might exert a negative effect on embryonic development and another approach should be needed for RNAi transgenesis in mice.

Characterization of Genomic Integration and Transgene Organization in Six Transgenic Rapeseed Events

To characterize the DNA rearrangement of both the T-DNA region and the genomic insertion site during T-DNA insertion, the Genomewalker strategy was used to isolate the junctions between the inserted DNA and the plant genomic DNA in six rapeseed events as well as the genomic DNA at the sites before integration. During transformation in each of the six events, portions of both the right border（RB） and left border（LB） regions of the T-DNA were deleted, ranging from a 7 nucleotide deletion of the LB repeats in event RF1 to a 207 bp deletion of the LB region in event RF2. For the six events, T-DNA integration resulted in a deletion at the target site spanning less than 100 bp. Sequence analysis indicated that the T-DNA was integrated into the coding region of various native rapeseed genes in events RF1 and RF2. Duplications of the genomic DNA target site were observed in events RF2, RF3 and Topas 19/2. And multimerization of transgenes was found in event Topas 19/2, in which, the T-DNA was integrated as a head-to-head（RB-to-RB） concatemer into the recipient genome. In event MS1, chromosomal translocation or a large target-site deletion may have occurred during T-DNA integration, which was identified due to a failure to amplify the presumptive insertion site based on the flanking rapeseed DNA sequences. Our results provide comprehensive data concerning transgene organization and the genomic context of the T-DNA in six rapeseed events, which can aid in the developing of insert fingerprinting and the monitoring of long-term genetic stability and potential unintended effects of transgenic events.

Establishment of a developmental atlas and transgenetic tools in the ascidian Styela clava

The ascidian Styela clava is an ecologically important species that is distributed along coastal regions worldwide.It has a long history as a model animal for evolutionary and developmental biology research owing to its phylogenetic position between vertebrates and invertebrates,and its classical mosaic expression patterns.However,the standard developmental atlas and protocols and tools for molecular manipulation of this organism are inadequate.In this study,we established a standard developmental table and provided a web-based digital image resource for S.clava embryogenesis at each developmental stage from fertilized eggs to hatching larvae by utilizing confocal laser microscopy and 3D reconstruction images.It takes around 10 h for fertilized eggs to develop into swimming larvae and 20–30 min to complete the tail regression processes at the metamorphic stage.We observed that the notochord cells in S.clava embryos did not produce an extracellular lumen like Ciona robusta,but showed polarized elongation behaviors,providing us an ideal comparative model to study tissue morphogenesis.In addition,we established a chemical-washing procedure to remove the chorion easily from the fertilized eggs.Based on the dechorionation technique,we further realized transgenic manipulation by electroporation and successfully applied tissue-specific fluorescent labeling in S.clava embryos.Our work provides a standard imaging atlas and powerful genetic tools for investigating embryogenesis and evolution using S.clava as a model organism.

Wheat kinase TaSnRK2.4 forms a functional module with phosphatase TaPP2C01 and transcription factor TaABF2 to regulate drought response

SNF1-related protein kinase 2(SnRK2)family members are essential components of the plant abscisic acid(ABA)signaling pathway initiated by osmotic stress and triggering a drought stress response.This study characterized the molecular properties of TaSnRK2.4 and its function in mediating adaptation to drought in Triticum aestivum.Transcripts of TaSnRK2.4 were upregulated upon drought and ABA signaling and associated with drought-and ABA-responsive cis-elements ABRE and DRE,and MYB and MYC binding sites in the promoter as indicated by reporter GUS protein staining and activity driven by truncations of the promoter.Yeast two-hybrid,BiFC,and Co-IP assays indicated that TaSnRK2.4 protein interacts with TaPP2C01 and an ABF transcription factor(TF)TaABF2.The results suggested that TaSnRK2.4 forms a functional TaPP2C01-TaSnRK2.4-TaABF2 module with its upstream and downstream partners.Transgene analysis revealed that TaSnRK2.4 and TaABF2 positively regulate drought tolerance whereas TaPP2C01 acts negatively by modulating stomatal movement,osmotic adjustment,reactive oxygen species(ROS)homeostasis,and root morphology.Expression analysis,yeast one-hybrid,and transcriptional activation assays indicated that several osmotic stress-responsive genes,including TaSLAC1-4,TaP5CS3,TaSOD5,TaCAT1,and TaPIN4,are regulated by TaABF2.Transgene analysis verified their functions in positively regulating stomatal movement(TaSLAC1-4),proline accumulation(TaP5CS3),SOD activity(TaSOD5),CAT activity(TaCAT1),and root morphology(TaPIN4).There were high correlations between plant biomass and yield with module transcripts in a wheat variety panel cultivated under drought conditions in the field.Our findings provide insights into understanding plant drought response underlying the SnRK2 signaling pathway in common wheat.

Barley chitinase genes expression revamp resistance against whitefly (Bemisia Tabaci) in transgenic cotton (Gossypium hirsutum L.)

Background Chitinase is an enzyme that hydrolyzes chitin,a major component of the exoskeleton of insects,including plant pests like whiteflies.The present study aimed to investigate the expression of chemically synthesized barley ch1 and chi2 genes in cotton(Gossypium hirsutum)through Agrobacterium-mediated transformation.Fifty-five putative transgenic cotton plants were obtained,out of which fifteen plants successfully survived and were shifted to the field.Using gene-specific primers,amplification of 447 bp and 401 bp fragments confirmed the presence of the ch1 and chi2 genes in five transgenic cotton plants of the T0 generation.These five plants were further evalu-ated for their mRNA expression levels.The T0 transgenic cotton plants with the highest mRNA expression level and better yield performance in field,were selected to raise their subsequent progenies.Results The T1 cotton plants showed the highest mRNA expression levels of 3.5-fold in P10(2)for the ch1 gene and 3.7-fold in P2(1)for the chi2 gene.Fluorescent in situ hybridization(FISH)confirmed a single copy number of ch1 and chi2(hemizygous)on chromosome no.6.Furthermore,the efficacy of transgenes on whitefly was evaluated through an insect bioassay,where after 96 h of infestation,mortality rates of whitefly were calculated to be 78%–80%in transgenic cotton plants.The number of eggs on transgenic cotton plants were calculated to be 0.1%–0.12 per plant compared with the non-transgenic plants where egg number was calculated to be 0.90–1.00 per plant.Conclusion Based on these findings,it can be concluded that the chemically synthesized barley chitinase genes(ch1 and chi2)have the potential to be effective against insects with chitin exoskeletons,including whiteflies.The transgenic cotton plants expressing these genes showed increased resistance to whiteflies,resulting in reduced egg numbers and higher mortality rates.

Integration mechanisms of transgenes and population fitness of GH transgenic fish

It has been more than 20 years since the first batch of transgenic fish was produced. Five stable germ-line transmitted growth hormone (GH) transgenic fish lines have been generated. This paper reviews the mechanisms of integration and gene targeting of the transgene, as well as the viability, reproduction and transgenic approaches for the reproductive containment of GH-transgenic fish. Further, we propose that it should be necessary to do the following studies, in particularly, of the breeding of transgenic fish: to assess the fitness of transgenic fish in an aqueous environment with a large space and a complex structure; and to develop a controllable on-off strategy of reproduction in transgenic fish.

Sustained high level transgene expression in mammalian cells mediated by the optimized piggyBac transposon system

Sustained,high level transgene expression in mammalian cells is desired in many cases for studying gene functions.Traditionally,stable transgene expression has been accomplished by using retroviral or lentiviral vectors.However,such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression.The piggyBac transposon has emerged as a promising nonviral vector system for efficient gene transfer into mammalian cells.Despite its inherent advantages over lentiviral and retroviral systems,piggyBac system has not been widely used,at least in part due to their limited manipulation flexibilities.Here,we seek to optimize piggyBac-mediated transgene expression and generate a more efficient,user-friendly piggyBac system.By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase(PBase),we demonstrate that adenovirusmediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells,compared to that obtained from co-transfection of the CMV-PBase plasmid.We further determine the drug selection timeline to achieve optimal stable transgene expression.Moreover,we demonstrate that the transgene copy number of piggyBac-mediated integration is approximately 10 times higher than that mediated by retroviral vectors.Using the engineered tandem expression vector,we show that three transgenes can be simultaneously expressed in a single vector with high efficiency.Thus,these results strongly suggest that the optimized piggyBac system is a valuable tool for making stable cell lines with sustained,high transgene expression.