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.

Application of transgenic mice to the molecular pathogenesis of cataract

One of the most prevalent disorders that cause blindness worldwide is cataract,and its essence is the visual disorder caused by the opacity of the lens.The significant degree of variation in cataracts and the fact that a variety of factors can impact a patient’s lens transparency make it especially crucial to investigate the pathogenesis of cataracts at the molecular level.It has been found that more than 60 genes are linked to the formation of cataracts,and the construction of a transgenic mouse model of cataract similar to the selection of human lens clouding due to a variety of causes has become an important means of studying the pathogenesis of cataract.Therefore,the research on the application of transgenic mice to the molecular pathogenesis of cataracts will be the main topic of this review of the literature.

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.

Fast,simple,efficient Agrobacterium rhizogenes-mediated transformation system to non-heading Chinese cabbage with transgenic roots

Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin.The one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.

c-Ha-ras在转基因小鼠模型C57-ras各组织中的表达谱分析

目的:分析转基因c-Ha-ras在C57-ras癌症小鼠模型中的组织表达谱及时空表达差异。方法:利用半定量及荧光定量RT-PCR法,分析不同首建鼠系、不同周龄小鼠各脏器中转基因c-Ha-ras的表达。结果:转基因c-Ha-ras在心、肝等13种组织器官中均有表达,在肺脏中表达最高,在肝脏中表达最低,No.2、No.3和No.5等3个首建鼠均呈现相似的变化规律;转基因在No.5首建鼠中表达水平最高,而在同一个首建鼠系中,12周龄时表达高于8周龄和24周龄。结论:转基因c-Ha-ras在各脏器中能高效表达,并间接表明该转基因能稳定遗传,为C57-ras癌症小鼠模型用于新药临床前致癌性评价提了供理论支持。

利用荧光定量PCR分析c-Ha-ras基因在C57-ras转基因小鼠中的拷贝数

目的:利用real-time PCR建立检测C57-ras转基因小鼠中外源c-Ha-ras基因拷贝数的简便方法,为药物安全性评价C57-ras转基因小鼠模型的繁殖和筛选提供数据支持。方法:以自建的人原癌基因c-Ha-ras转基因小鼠为研究对象,利用SYBR GreenⅠ荧光定量PCR的绝对定量法测定3个C57-ras转基因小鼠系的c-Ha-ras基因Ct值,通过与内参基因GAPDH比较计算获得转基因的拷贝数。结果:内参基因GAPDH的标准曲线为lg NGAPDH=-2.852Ct+26.236,外源基因c-Ha-ras的标准曲线为lg NRAS=-3.068Ct+39.186;经计算,NO.2、NO.3、NO.5系的F6代拷贝数分别为4、4和3,并且系内不同个体间拷贝数一致。结论:利用SYBR GreenⅠreal-time PCR技术建立了检测C57-ras转基因小鼠中外源基因拷贝数的方法,该方法操作简单,节约成本,为C57-ras致癌性模型的选留和应用提供了基础和技术手段,也为其他类似转基因品系中转基因拷贝数的确定提供了一种参考方法。

c-Ha-ras转基因小鼠的构建与鉴定

采用 ＰＣＲ和定点突变的方法获得 ｃ－Ｈａ－ｒａｓ突变基因，将其克隆到ｐｃＤＮＡ３载体上，得到含有 ｃ Ｈａ－ｒａｓ６１密码子突变和核苷酸 ２７１３突变的质粒 ｐｃＤＮＡ３－ｒａｓ２。对重组质粒进行瞬时表达，Ｗｅｓｔｅｒｎ Ｂｌｏｔ检测到强阳性条带。酶切重组质粒，回收含有ＣＭＶ启动子－目的基因－下游调控序列的４．３ｋｂ片段，然后将其显微注射到１ＣＲ小鼠受精卵中。将注射后的受精卵移入假孕母鼠的输卵管进行母体孕育。小鼠出生３周后，ＰＣＲ和Ｓｏｕｔｈ－ｅｒｎ Ｂｌｏｔ检测证实有４只小鼠基因组中整合了外源ｃ－Ｈａ－ｒａｓ突变基因。上述结果表明已成功构建了 ４只 ｃ－Ｈａ－ｒａｓ转基因小鼠。

人肺癌组织中C-Ha-ras,C-myc和C-sis基因的扩增

用同位素ａ－３２ＰｄＣＴＰ标记癌基因Ｃ－Ｈａ－ｒａｓ，Ｃ－ｍｙｃ和Ｖ－ｓｉｓ基因片段，通过点杂交和Ｓｏｕｔｈｅｒｎ杂交法检测１７例鳞癌，９例腺癌，６例其它类型肺癌组织和６例非肺癌组织中Ｃ－Ｈａ－ｒａｓ，Ｃ－ｍｙｃ和Ｃ－ｓｉｓ基因扩增的情况．结果发现：４６．８％（１５／３２）的肺癌组织有Ｃ－Ｈａ－ｒａｓ基因扩增，其中１０例（５８．８ ％）鳞癌，３例腺癌（３３．３ ％）出现Ｃ－Ｈａ－ｒａｓ基因扩增。２５％（８／３２）的肺癌组织中出现Ｃ－ｍｙｃ基因扩增，其中 ４例（２３．５％）鳞癌，３例（３３．３％）腺癌出现Ｃ－ｍｙｃ基因扩增。１８．８％（６／３２）的肺癌组织有Ｃ－ｓｉｓ基因扩增，其中４例（２３．５％）鳞癌，１例（１１．１％）腺癌出Ｃ－ｓｉｓ基因扩增。而６例非肺癌组织都役有这三种癌基因扩增。同时我们也发现有５例肺癌组织（４例鳞癌和１例腺癌）出现两种癌基因的扩增。上述结果提示：Ｃ－Ｈａ－ｒａｓ，Ｃ－ｍｙｃ和Ｃ－ｓｉｓ基因的扩增与肺癌的发生有关，特别是Ｃ－Ｈａ－ｒａｓ基因扩增在肺鳞癌的形成过程中可能起着重要作用。同时还提示：肺癌的发生与两种癌基因的扩增可能有一定关系。

p53和C-Ha-ras基因在胃癌发生中的作用

目的 探讨 p5 3和C Ha ras基因突变在胃癌发生中的作用。 方法 用PCR SSCP和直接测序检测胃癌组织中C Ha ras(12、13密码子 )和 p5 3(外显子 7)基因的突变。 结果 胃癌组织中C Ha ras和 p5 3基因突变发生率显著高于浅表性胃炎和正常胃黏膜组织 ,基因的联合突变在部分胃癌组织中表达。结论 基因联合突变可能是胃癌发生的机制之一。

亚硒酸钠对人胃腺癌细胞中c-Ha-ras和-cerbB2癌基因及其蛋白的影响

应用细胞原位杂交和免疫胶体金技术，观察亚硒酸钠处理人胃腺癌细胞前后，ｃ－Ｈａ－ｒａｓ和－ｃｅｒｂＢ２癌基因转录水平及其产物Ｐ２１和Ｐ１８５癌蛋白表达的变化，结果表明，两种癌基因在ＭＧｃｘ８０－３细胞中活跃表达，亚硒酸钠能抑制ｃ－Ｈａ－ｒａｓ和－ｃｅｒｂＢ２癌基因的转录水平及Ｐ２１和Ｐ１８５癌蛋白的表达，这对于诱导胃癌细胞向正常细胞方向逆转具有重要意义。

Impact of Copy Number on Transgene Expression In Tobacco

对农杆菌介导法获得的转 β_葡糖醛酸酶 (β_glucuronidase ,GUS)基因 (uidA)烟草 (NicotianatabacumL .)进行GUS表达分析 ,发现部分转基因植株无GUS活性。进一步Southern杂交结果发现 ,GUS基因失活植株的基因组中整合了多个uidA拷贝 ,而GUS活性高的转基因植株多为uidA单拷贝整合 ,表明uidA基因失活与基因多拷贝整合有关。Northern杂交结果显示 ,失活植株无特异uidARNA杂交带 ,而GUS活性高的植株可检测到明显的杂交信号 ,说明多拷贝引起的基因失活发生在RNA水平。

大鼠肝癌变过程中几种癌基因的原位表达及c-Ha-ras1点突变的研究

目的 研究大鼠肝癌变过程中几种癌基因的表达和c Ha ras 1点突变。方法 通过DEN诱发癌变启动模型和肝癌模型 ,应用免疫组化、原位杂交和MDT PCR SSCP分别观测癌基因的表达和c Ha ras1点突变。结果 c Ha ras和c myc基因的过表达参与了肝癌变的全过程 ,其中c Ha ras的过表达与癌前嗜碱性肝细胞灶、c myc基因的过表达与卵圆细胞增殖关系密切。IGF Ⅱ基因的过表达对癌前肝细胞增生灶 /结节向肝细胞癌的演进起重要作用。fms基因过表达仅与个别大鼠肝细胞癌有关。结论 大鼠肝癌变过程与c Ha ras、c myc、IGF Ⅱ和fms基因的过表达及c Ha ras1点突变有关 。

妊娠滋养细胞疾病c-Ha-ras基因突变及ras p21蛋白表达的研究

目的 :探讨c -Ha -ras基因突变及 p21蛋白表达与妊娠滋养细胞疾病 (GTD)发生发展的关系 ,寻找具有特异性的可预测PTD的诊断标记。方法 :采用PCR -SSCP法及SP免疫组化法 ,对90例GTD组织中c -Ha -ras基因第1外显子点突变及其产物 p21表达进行检测 ,以正常足月新鲜胎盘30例为对照。结果 :所有正常胎盘组织ras突变及p21表达均为阴性。ras基因突变在恶性GTD中为36 7 % ,CM将来发展成为PTD的为43 3 % ,均高于CM无PTD发展的16 7 %。恶性GTD,Ⅰ、Ⅱ、Ⅲ级组织分化者点突变率分别为15 4 %、40 %、71 4 % ,各级间差异有显著性 (P<0 01)。不同转归的CM,p21表达不同 ,CM中发展成为PTD者 ,p21表达率为83 3 % ,明显高于未发展成PTD者的43 3 %(P<0 005) ,亦高于恶性GTD的60%(P<0 05)。结论 :GTD的发展演进与ras基因突变及p21表达有关 ,ras突变及p21表达可考虑作为预测PTD的诊断指标之一。GTD由良性到恶性的发展过程中 ,p21表达量存在由低→高→低的内在变化 。

Influence of Matrix Attachment Regions from Maize on Transgene Expression Level in Tobacco

The effect of matrix attachment regions (MARs) on foreign gene expression in transgenic plants was studied, The beta-glucuronidase (GUS) gene (uidA) was flanked by the MARs isolated from the genome of maize to form plant expression vector. The vectors with and without MARs were transferred into tobacco ( Nicotiana tabacum L.) through Agrobacterium-mediated transformation procedure. GUS activity assays indicated that MARs could increase expression level of uidA gene. The mean GUS activity could be increased twofold as compared to that of transformants without MARs, and the highest GUS activity of transformant could reach tenfold. The correspondence between GUS activity and mRNA accumulation was positive and indicated that MARs could improve transcription of foreign gene.

C-Ha-ras基因突变与妊娠滋养细胞疾病发生、发展及恶变的关系

应用分子生物学ＰＣＲ－ＳＳＣＰ法对正常足月新鲜胎盘３０例，葡萄胎（ＨＭ）终止后发展成持续性滋养细胞病（ＰＴＤ）３０例，未发展成持续性滋养细胞病（非ＰＴＤ）３０例，恶性滋养细胞疾病（ＧＴＤ）３０例Ｃ－Ｈａ－ｒａｓ基因点突变进行检测。结果：正常足月胎盘无Ｃ－Ｈａ－ｒａｓ基因点突变；在恶性ＧＴＤ中Ｃ－Ｈａ－ｒａｓ基因突变的阳性率３６．７％，ＰＴＤ中阳性率４３．３％，非ＰＴＤ中阳性率１６．７％。提示：Ｃ－Ｈａ－ｒａｓ基因点突变与ＧＴＤ的发生、发展和恶性演变有关；阳性率随组织学分级差而升高，反映恶性ＧＴＤ的恶性程度。

葡萄胎组织中c-Ha-ras基因突变的初步研究

目的:为了在分子水平了解c-Ha-ras基因在葡萄胎疾病中的变化,寻找预测葡萄胎恶变的途径。方法:用聚合酶链反应-限制性片段长度多态性分析(PCR-RFLP)法测定恶变与非恶变组葡萄胎组织中c-Ha-ras基因突变的情况。结果:c-Ha-ras第12位密码子的突变率恶变组为60.0％,未恶变组为26.7％,二者具有显著性差异(P<0.05)。在正常绒毛组织中无1例恶变。结论:c-Ha-ras第12位密码子的突变可能与葡萄的恶变率增高有关。

应用聚合酶链反应直接测序检测C-Ha-ras基因突变

建立一种新的检测C-Ha-ras基因突变的方法—聚合酶链反应产物直接测序,此法可识别C-Ha-ras基因突变的部位和特性,由于应用Taq DNA聚合酶和热循环装置,所需模板DNA量明显减少,以及采用^(32)P同位素末端标记引物,过夜爆光就可以显示模板DNA序列,X线片上的序列带单一,背景低,结果准确.

宫颈癌中C-Ha-ras癌基因点突变的研究

宫颈癌是妇女生殖系统最常见的恶性肿瘤,严重威胁着我国妇女的身体健康和生命安全,对宫颈癌癌基因的研究有助于宫颈癌的早期诊断、防治以及肿瘤的分型。我们在国内首次将体外基因扩增技术(即PCR技术)应用于宫颈癌癌基因的研究,先人工合成两个特异性的寡核苷酸引物,在耐热DNA多聚酶的催化下,将宫颈癌DNA中C-Ha-ras癌基因第一个外显子的核苷酸序列放大百万倍左右,再以同位素标记的特异寡核苷酸探针杂交。