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

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Zoological Research 2024年第3期45卷 567-574页

作者： Peng-Cheng Wang Hao Deng Rang Xu Jiu-Lin Du Rongkun Tao Department of Pediatric Cardiology Xinhua HospitalShanghai Jiao Tong University School of MedicineShanghai 200092China Institute of Neuroscience State Key Laboratory of NeuroscienceCenter for Excellence in Brain Science and Intelligence TechnologyChinese Academy of SciencesShanghai 200031China Medical School Kunming University of Science and TechnologyKunmingYunnan 650500China Scientific Research Center Xinhua HospitalShanghai Jiao Tong University School of MedicineShanghai 200092China University of Chinese Academy of Sciences Beijing 100049China School of Life Science and Technology ShanghaiTech UniversityShanghai 200031China

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.

关键词： Zebrafish Tol2 transposase transgene Large payload Synthetic biology

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

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Journal of Cotton Research 2024年第1期7卷 90-100页

作者： BASHIR Samina YAQOOB Amina BASHIR Rohina BUKHARI Shanila SHAHID Naila AZAM Saira BAKHSH Allah HUSNAIN Tayyab SHAHID Ahmad Ali RAO Abdul Qayyum Centre of Excellence in Molecular Biology University of the PunjabLahore 53700Pakistan Department of Botany Kinnaird College for Women UniversityLahore 54000Pakistan Department of Botany Government College UniversityFaisalabad 38000Pakistan Institute of Botany University of the PunjabLahore 54590Pakistan.

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.

关键词： Chitinase Cotton White fly transgene Bioassay

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

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Rice science 2019年第2期26卷 109-117,I0016,I0017页

作者： HE Yubing ZHU Min WANG Lihao WU Junhua WANG Qiaoyan WANG Rongchen ZHAO Yunde National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan) Huazhong Agricultural University College of Life Science and Technology Huazhong Agricultural University College of Plant Science & Technology Huazhong Agricultural University Section of Cell and Developmental Biology University of California San Diego

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.

关键词： genome editing suicide gene transgene killer CRISPR Cas9 transgene-free

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Influence of DNA methylation on transgene expression

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Chinese Science Bulletin 2001年第15期46卷 1300-1304页

作者： Xugang Li Zhen Zhu Dejiang Feng Tuanjie Chang Xiang Liu Chinese Acad Sci Inst Genet Beijing 100101 Peoples R China

DNA methylation plays an important role in gene expression in eukaryote. But DNA methylation of transgene usually leads to target gene silencing in plant genetic engineering. In this research, reporter gene β-glu-curonidase (GUS) gene (uidA) was introduced into tobaccos via Agrobacterium-mediated transformation method, and the foreign uidA gene became inactive in some transgenic tobaccos. No mRNA of uidA was detected in these plants by Northern blotting analysis, and DNA methylation of promoter region was found. The results indicated that gene silencing might be caused by DNA methylation of promoter.

关键词： DNA methylation gene silencing transgene GUS tran-scriptional product.

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Efficient selection of homozy-gous lines of hybrid rice restorer with the transgene Xa21 using test cross and PCR

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Chinese Science Bulletin 2001年第2期46卷 125-129页

作者： MA Bojun WANG Wenming ZOU Junhuang LIU Guoqing FANG Jin ZHU Lihuang ZHAI Wenxue Institute of Genetics Chinese Academy of SciencesBeijing 100101China Zhejiang Normal University Jinhua 321004China Yangzhou University Yangzhou 225009China

The homozygous restorer lines with a single copy of the transgene Xa21 have been obtained from the progenies of transgenic Minghui63 and Yanhui559 plants through PCR marker-assisted selection and test cross. These hom... 详细信息

关键词： test cross PCR marker-assisted selection rice restorer with the transgene Xa21 homozygous.

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Characterization of genetic humanized mice with transgenic HLA DP401 or DRA but deficient in endogenous murine MHC classⅡgenes upon Staphylococcus aureus pneumonia

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Animal Models and Experimental Medicine 2023年第6期6卷 585-597页

作者： Feng Li Bowen Niu Lingling Liu Mengmin Zhu Hua Yang Boyin Qin Xiuhua Peng Lixiang Chen Chunhua Xu Xiaohui Zhou Department of Laboratory Animal Science Shanghai Public Health Clinical CenterShanghaiChina

Background:Staphylococcus aureus can cause serious infections by secreting many superantigen exotoxins in“carrier”or“pathogenic”states.HLA DQ and HLA DR humanized mice have been used as a small animal model to study the role of two molecules during S.aureus infection.However,the contribution of HLA DP to S.aureus infection is unknown yet.Methods:In this study,we have produced HLA DP401 and HLA DRA0101 humanized mice by microinjection of C57BL/6J zygotes.Neo-floxed IAβ+/-mice were crossbred with Ella-Cre and further crossbred with HLA DP401 or HLA-DRA0101 humanized mice.After several rounds of traditional crossbreeding,we finally obtained HLA DP401-IAβ-/-and HLA DRA-IAβ-/-humanized mice,in which human DP401 or DRA0101 molecule was introduced into IAβ-/-mice deficient in endogenous murine MHC classⅡmolecules.A transnasal infection murine model of S.aureus pneumonia was induced in the humanized mice by administering 2×108CFU of S.aureus Newman dropwise into the nasal cavity.The immune responses and histopathology changes were further assessed in lungs in these infected mice.Results:We evaluated the local and systemic effects of S.aureus delivered intranasally in HLA DP401-IAβ-/-and HLA DRA-IAβ-/-transgenic mice.S.aureus Newman infection significantly increased the m RNA level of IL 12p40 in lungs in humanized mice.An increase in IFN-γand IL-6 protein was observed in HLA DRA-IAβ-/-mice.We observed a declining trend in the percentage of F4/80+macrophages in lungs in HLA DP401-IAβ-/-mice and a decreasing ratio of CD4+to CD8+T cells in lungs in IAβ-/-mice and HLA DP401-IAβ-/-mice.A decreasing ratio of Vβ3+to Vβ8+T cells was also found in the lymph node of IAβ-/-mice and HLA DP401-IAβ-/-mice.S.aureus Newman infection resulted in a weaker pathological injury in lungs in IAβ-/-genetic background mice.Conclusion:These humanized mice will be an invaluable mouse model to resolve the pathological mechanism of S.aureus pneumonia and study what role DP molecule plays in S.aureus infection.

关键词： HLA DP401 HLA-DRA humanized mice MHC II Staphylococcus aureus pneumonia transgene

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Production of marker-free transgenic plants from mature tissues of navel orange using a Cre/loxP site-recombination system

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Horticultural Plant Journal 2023年第3期9卷 473-480页

作者： Yongrui He Lanzhen Xu Aihong Peng Tiangang Lei Qiang Li Lixiao Yao Guojin Jiang Shanchun Chen Zhengguo Li Xiuping Zou Key Laboratory of Plant Hormones and Development Regulation of Chongqing School of Life SciencesChongqing UniversityChongqing 401331China National Citrus Engineering Research Center(NCERC) Citrus Research InstituteSouthwest University/Chinese Academy of Agricultural SciencesChongqing 400712China

Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciensmediated transformation for generation of marker-free transgenic plants from navel orange(Citrus sinensis Osbeck) mature stems using a CreloxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1-0.5(1-2 weeks), > 0.5 cm(3-4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange(Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase(IPT) and an anti-bacterial peptide(PR1aCB).Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained.Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants(deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange(Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.

关键词： Citrus sinensis Osbeck Mature stem segment Marker-free transgene Bud micrografting

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

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Oil Crop Science 2018年第2期3卷 92-98页

作者： Shouwei Zhang Shi Sun Jing Sun Xiujie Liu Wensheng Hou Tingting Wu Cunxiang Wu Tianfu Han Bingjun Jiang MOA Key Laboratory of Soybean Biology (Beijing) Institute of Crop ScienceThe Chinese Academy of Agricultural Sciences

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.

关键词： genetically modified soybean high methionine transgene insertion identification,next generation sequencing

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Effects of donor cells on in vitro development of cloned bovine embryos

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Journal of Genetics and Genomics 2008年第5期35卷 273-278页

作者： Jing Fu Pengfei Guan Leiwen Zhao Hua Li Shuzhen Huang Fanyi Zeng Yitao Zeng Shanghai Institute of Medical Genetics Shanghai Children＇s Hospital Shanghai Jiao Tong University School of Medicine Shanghai 200040 China

The donor cells from different individuals and with different foreign genes introduced were investigated to determine their effects on the efficiency of somatic cell nuclear transfer （SCNT）. The bovine ear fibroblast from different individuals was isolated, cultured, and then transfected with foreign genes to establish the stable cell lines, which were used as donor cells for nuclear transfer. The oocytes were obtained through ovum pick up operation. After in vitro maturation, the M II phase oocytes were selected as receptors for nuclear transfer. The reconstructed embryos were cultured in vitro and observed at 2 h, 48 h, and 7 days after transfer to assess the rate of fusion using cleaved and blastocyst as the parameters of SCNT efficiency. The donor cells from different individuals （04036, 06081, 06088, and 06129） had no obvious effect on the fusion and cleaved rate, whereas there was significant difference in the blastocyst rate （P〈0.05）, and the rate was 62.3%, 37.0%, 35.1%, and 15.6%, respectively. There was no significant difference among the rate of fusion, cleaved and blastocyst in donor cells with different foreign genes （P〉0.05）. It was concluded that the genetic background of the donor cells could affect the efficiency of SCNT, while the introduction of foreign genes into the donor cells had no obvious effect on the efficiency. This study provides useful information for the SCNT and would benefit in promoting the efficiency.

关键词： donor cell bovine somatic nuclear transfer transgene

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Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions

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Cell Research 2007年第1期17卷 62-72页

作者： Betty Ying Zhou Prashant Mali USAThe Institute for Cell Engineering Baltimore Whitehead institute for Biomedical Research MD Program of Biomedical Engineering the Johns Hopkins UniversiO MD 21205.USA. The Institute for Cell Engineering Department of Gynecology & Obstetrics the Johns Hopkins University School of Medicine MA 02142 Cambridge

We previously showed that Wnt3a could stimulate human embryonic stem (hES) cell proliferation and affect cell fatedetermination.In the absence of feeder cell-derived factors,hES cells cultured under a feeder-free condition survivedand proliferated poorly.Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cellproliferation but also differentiation.In the present study,we further extended our analysis to other Wnt ligands such asWntl and Wnt5a.While Wntl displayed a similar effect on hES cells as Wnt3a,Wnt5a had little effect in this system.Wnt3a and Wntl enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors andbFGF are also present.To explore the possibility to promote the proliferation of undifferentiated hES cells by activatingthe Wnt signaling,we overexpressed Wnt3a or Wntl gene in immortalized human adult fibroblast (HAFi) cells that aresuperior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts.HAFicells with or without a Wnt transgene can be propagated indefinitely.Over-expression of the Wnt3a gene significantlyenhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested.Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us toselect rare hES clones after stable transfection or transduction.These immortalized engineered feeder cells (W3R) thatco-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently makegenetic modified hES cell lines for basic and translational research.

关键词： WNT human embryonic stem cells stem cell renewal stem cell differentiation transgene

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