显微注射法制备遗传工程小鼠模型的研究

遗传工程小鼠模型是基因功能、人类疾病发病机制及新药研究开发的最重要的模式生物之一。在建立遗传工程小鼠模型的众多方法中,显微注射法是目前国际上公认的制备转基因及基因剔除动物模型的首选。在进行了大量显微注射工作后,简要介绍建立遗传工程小鼠模型的基本技术与方法,并对在显微操作过程中较为关键的因素进行一些分析和讨论。     

前列腺特异启动子和部分靶基因在转基因模型中的应用进展

遗传工程小鼠模型已被大量用于前列腺癌发生、发展、恶化和转移的研究。构建小鼠模型的途径主要包括转基因的过表达、基因敲除或敲入,以及使用Cre/lox技术进行条件调控。本文综述了目前构建的有明显遗传表型的转基因小鼠模型所用到的前列腺特异的启动子,以及所用到的靶基因。     

通过诱导剂作用于启动子的条件性基因表达

通过遗传工程技术获得的转基因动植物对分析某些生化过程和发育途径极为有用。通过化学诱导剂作用于启动子的条件性基因表达是分子生物学和生物技术应用研究中的强有力的手段。建立于目标基因激活和失活基础之上的几个究子诱导基因表达系统已有报道。将来自于原核生物、昆虫和其它动物的调节因子应用于新的物种有利于促进转基因技术的应用和有关基因的时空表达研究。本文综述了有关的基因表达调节系统,启动子激活的基因表达系统,启动子失活的基因表达系统,以及可诱导的基因过度表达和反义抑制系数。     

通过诱导剂作用于启动子的条件性基因表达(英文)

通过遗传工程技术获得的转基因动植物对分析某些生化过程和发育途径极为有用。通过化学诱导剂作用于启动子的条件性基因表达是分子生物学和生物技术应用研究中的强有力的手段。建立于目标基因激活和失活基础之上的几个化学分子诱导基因表达系统已有报道。将来自于原核生物、昆虫和其它动物的调节因子应用于新的物种有利于促进转基因技术的应用和有关基因的时空表达研究。本文综述了有关的基因表达调节系统 ,启动子激活的基因表达系统 ,启动子失活的基因表达系统 ,以及可诱导的基因过度表达和反义抑制系统     

成年大脑神经细胞特异性ADAM10基因敲除小鼠模型的建立与鉴定

去整合素和金属蛋白酶10(ADAM10)是一种能够水解30余种跨膜蛋白质的“脱落酶”(sheddase), 参与诸多生理过程和致病机制, 如胚胎发育、细胞粘附、信号转导、免疫反应、癌症和阿尔茨海默病。迄今, 已报道的ADAM10完全基因敲除小鼠和大脑神经前体细胞特异性ADAM10基因敲除小鼠分别于胚胎期或围产期死亡, 致使无法研究成年小鼠大脑神经细胞ADAM10基因的功能。文章利用本研究小组建立的CaMKIIα-Cre转基因小鼠与ADAM10^loxP/loxP转基因小鼠杂交, 获得了CaMKIIα-Cre/ADAM10^loxP/loxP小鼠, 并对其进行鉴定。利用PCR方法检测成年ADAM10 cKO小鼠大脑基因组DNA表明, ADAM10基因缺失主要发生在前脑皮层和海马中。荧光定量PCR检测结果显示, ADAM10 mRNA的表达水平在前脑皮层和海马中分别降低55.7%和60.8% ; 使用Western blotting方法研究发现, ADAM10成熟蛋白质的含量在前脑皮层和海马中分别减少63%和84.8% 。采用免疫组织化学方法检测表明, 成年ADAM10 cKO小鼠与野生型小鼠相比, 其大脑皮层和海马神经细胞的ADAM10免疫染色明显减弱, 而其它细胞如胶质细胞的免疫染色基本一致。总之, 文章成功制备了首个存活至成年的大脑神经细胞特异性ADAM10基因敲除(cKO)小鼠, 克服了小鼠因ADAM10缺失在胚胎期或围产期死亡的弊端, 为研究成年小鼠大脑神经细胞ADAM10基因的功能奠定了坚实的基础。     

条件性基因敲除:骨髓细胞特异性敲除Ncol2基因小鼠的建立和基因型鉴定

Ncol2是新发现的参与免疫调节的重要因子,Ncol2基因骨髓细胞特异性敲除小鼠的建立,能够有针对性的研究Ncol2基因缺失后对免疫系统的影响。根据条件性基因敲除的原理,本文利用loxp转基因小鼠和在骨髓细胞特异性表达Cre重组酶的LysMcre小鼠,繁殖建立了骨髓细胞特异性敲除Ncol2基因的小鼠,并提供了用鼠尾做基因型鉴定的简便方法。     

斑马鱼leg1直系同源基因mu-leg1在小鼠中的表达谱分析

基因leg1(liver-enriched gene 1)首先在斑马鱼中作为肝脏富集表达基因被鉴定。进一步的研究揭示leg1编码的Leg1蛋白代表一类新型外分泌蛋白, 它在斑马鱼胚期肝脏生长发育过程中起关键作用。小鼠leg1(mu-leg1)是斑马鱼leg1(zb-leg1)的直系同源基因, 二者编码的蛋白氨基酸序列相似性为31%。文章通过巢式PCR从成年小鼠肝脏中成功克隆了mu-leg1的cDNA序列, 并对该基因在成年小鼠不同组织中的表达特征进行分析和鉴定。Northern印迹杂交和半定量RT-PCR分析结果显示, mu-leg1在成年小鼠小肠中而非肝脏中富集表达。此外, 用制备的mu-Leg1多克隆抗体进行Western印迹杂交, 结果显示mu-Leg1也是一个分泌蛋白。同时, 还建立了mu-leg1基因条件性剔除杂合子小鼠。这些材料为今后深入研究和探讨mu-Leg1蛋白的生化功能奠定了基础。     

骨形成蛋白4条件性RNA干扰小鼠的建立

为研究骨形成蛋白4(Bone morphogenetic protein 4, BMP4)在骨骼发育中的作用, 我们以含有LoxPneo的pBSK/U6载体为骨架, 构建小鼠BMP4条件性RNAi(conditional RNA interference), CRNA; 载体(BMP4CRNAi), 经KpnⅠ和AflⅢ双酶切获取针对bmp4并含neo基因的目的干扰片段, 纯化后的目的片段显微注射入0.5 d的FVB/NJ小鼠受精卵, 并植入同期发情的假孕母鼠中, 获取G0代转基因小鼠; 利用PCR对G0代转基因小鼠基因型进行鉴定, PCR阳性的小鼠与FVB/NJ小鼠交配, 最终获取稳定传代的BMP4CRNAi小鼠。稳定传代的BMP4^CRNAi小鼠与成骨和软骨前体细胞表达Cre的转基因小鼠(Col2a1-Cre)交配, 获取BMP4^Col2a1-CRNAi小鼠。分离BMP4Col2a1-CRNAi小鼠原代软骨细胞, 提取总RNA, 利用半定量RT-PCR检测RNA干扰效率。小鼠基因型鉴定结果表明：成功获得条件性RNAi转基因小鼠; BMP4干扰效率检测结果表明：在软骨细胞中BMP4的干扰效率为81％。以上结果表明, 我们成功制备了BMP4^CRNAi小鼠和BMP4^Col2a1-CRNAi小鼠, BMP4^CRNAi小鼠与不同Cre转基因小鼠交配, 可以研究BMP4在不同细胞、组织和器官的功能, BMP4^Col2a1-CRNAi小鼠的获得为研究BMP4在软骨发育中的作用提供了合适的动物模型。     

GPR126条件性基因敲除载体的快速构建

基因敲除小鼠模型是在动物体内研究基因功能的重要和可靠的手段之一,而构建用于同源重组的基因敲除载体是构建基因敲除小鼠模型的关键一步.条件性基因敲除技术将针对靶基因的修饰作用限制在小鼠的特定发育时期或特定类型的组织细胞中,通过加入具有位点特异性的重组系统,该修饰作用在时间和空间上均处于可调控的状态;条件性基因敲除技术既可以避免某些具有重要功能基因的敲除所带来的早期胚胎致死问题,还可用于精确分析某些多效基因在不同组织或不同发育阶段的特定功能差异.通过合理利用改良的Red重组系统,将两个LoxP位点快速准确地插入到了目标位置,实现了GPR126条件性基因敲除载体的快速构建,为后续的构建GPR126基因敲除小鼠模型、在动物体内研究基因的功能奠定了基础.     

Blood杂志发表健康所最新研究成果

palladin是一个伴随着NB4细胞在全反式维甲酸诱导下分化过程而表达上调的基因。几年前由健康科学研究所王铸钢研究员领导的遗传工程实验室构建了palladin缺失的小鼠模型,发现palladin的缺失导致小鼠胚胎致死同时伴有脑部神经管和腹壁的闭合障碍。最近该研究组的最新研究表明palladin缺失的小鼠胚胎在死亡之前有严重的贫血表型。     

基因工程微生物生态学研究进展

基因工程微生物(genctically enginccred microorganism,GEM)生态学的研究已成为微生物分子生态学的一项主要研究内容之一．随着分子标记和分子生物学检测手段的引入,传统的微生物生态学研究被注入了新的活力,在分子水平上探讨基因工程微生物与环境及环境中土著生物之间的关系已成为可能．基因工程微生物生态学是一门内容涉及分子生物学、微生物学、生态学等诸多学科的新型交叉边缘学科．本文提出加紧进行转基因生物生态学和转基因生物的风险评价的研究工作,建立适合中国国情的检测手段和评价标准,有助于我国基因工程微生物生态学的健康发展．     

Field applications of genetically engineered microorganisms for bioremediation processes

Genetically engineered microorganisms (GEMs) have shown potential for bioremediation applications in soil, groundwater, and activated sludge environments, exhibiting enhanced degradative capabilities encompassing a wide range of chemical contaminants. However, the vast majority of studies pertaining to genetically engineered microbial bioremediation are supported by laboratory-based experimental data. In general, relatively few examples of GEM applications in environmental ecosystems exist. Unfortunately, the only manner in which to fully address the competence of GEMs in bioremediation efforts is through long-term field release studies. It is therefore essential that field studies be performed to acquire the requisite information for determining the overall effectiveness and risks associated with GEM introduction into natural ecosystems.     

2018 iGEM专栏序言

国际基因工程机器大赛(iGEM),作为一项以合成生物学为主题,集合了多种交叉学科的学生科研赛事,已成为了当今生物科研领域属于年轻人的最具活力和影响力的舞台。近年来,许多来自国内的大学和高中队伍不仅在比赛中取得了优异成绩,还做出了具有创新性的科研成果。为此,我们特组织出版了此iGEM专栏,集中报道近年来国内多支iGEM参赛队的研究工作,同时关注、探讨iGEM大赛在中国的发展情况和对大学生科研能力培养的启示。     

实验小鼠在癌症研究中的应用及其进展

小鼠是生物医学研究中使用数量最多的哺乳类实验动物。人类利用小鼠模型进行癌症研究已有100多年的历史,小鼠大量的遗传变异可作为研究人类癌症的借鉴。特别是近年来,培育成功的转基因、基因敲除等遗传工程小鼠模型,使我们对人类癌症发生有了深刻的认识,为评估癌症的诊断方法,革新预防和治疗方案提供了一个很有价值的平台。本文着重介绍了癌症研究中常用的小鼠模型、GEM模型及取得的最新进展等,分析了小鼠肿瘤模型的局限性,并对其发展趋势进行展望。     

The strategy and potential utilization of temperate germplasm for tropical germplasm improvement: a case study of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

The organization of maize (Zea mays L.) germplasm into genetically divergent heterotic groups is the foundation of a successful hybrid maize breeding program. In this study, 94 CIMMYT maize lines (CMLs) and 54 United States germplasm enhancement of maize (GEM) lines were assembled and characterized using 1,266 single nucleotide polymorphisms (SNPs) with high quality. Based on principal component analysis (PCA), the GEM lines and CMLs were clearly separated. In the GEM lines, there were two groups classified by PCA corresponding to the heterotic groups “stiff stalk” and “non-stiff stalk”. CMLs did not form obvious subgroups by PCA. The allelic frequency of each SNP differed in GEM lines and CMLs. In total, 3.6% alleles (46/1,266) of CMLs are absent in GEM lines and 4.4% alleles (56/1,266) of GEM lines are absent in CMLs. The performance of F1 plants (n = 654) produced by crossing between different groups based on pedigree information was evaluated at the breeding nurseries of two CIMMYT stations. Genomic estimated phenotypic values of plant height and days to anthesis for a testing set of 45 F1 crosses were predicted based on the training data of 600 F1 crosses using a best linear unbiased prediction method. The prediction accuracy benefitted from the adoption of the markers associated with quantitative trait loci for both traits; however, it does not necessarily increase with an increase in marker density. It is suggested that genomic selection combined with association analysis could improve prediction efficiency and reduce cost. For hybrid maize breeding in the tropics, incorporating GEM lines which have unique alleles and clear heterotic patterns into tropically adapted lines could be beneficial for enhancing heterosis in grain yields.     

Special focus: Synthetic biology

Special focus: Synthetic biology What is synthetic biology? SynBERC – The Synthetic Biology Engineering Research Center Ars Synthetica iGEM – The International Genetically Engineered Machine competition Some synthetic biology companies Paper watch: Synthetic biology Building blocks for novel functions Knowledge-making distinctions in synthetic biology Scaffold design and manufacturing: From concept to clinic Peptidomimetics – a versatile route to biologically active compounds Metabolic engineering of E. coli E. coli needs safety valves Systems-level metabolic engineering Mammalian synthetic biology Chemical aspects of synthetic biology Synthesis of DNA fragments in yeast Synthetic biology and patentable subject matter Patenting artificial life? Metabolic effects of synthetic rewiring Engineering for biofuels Regulatory elements for synthetic biology Book highlight Systems Biology and Synthetic Biology     

Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.     

The new transgenic mouse core: no longer a facility just for making mice

The times they are a-changin' for GEM and the facilities that maintain them. Core facilities are expanding beyond their original conception as producers of transgenic mice to encompass a wide range of services, including research animal maintenance. In this paper, the authors describe the logistics and administration of the newly dubbed Mouse Genetics Core Facility at the Memorial Sloan-Kettering Cancer Center as a blueprint for other institutions seeking to expand and update their own transgenic cores for research in the twenty-first century.     

合成生物学与国际遗传工程机器大赛对培养大学生双创思维与能力的影响

合成生物学是21世纪前沿交叉学科,是现代生物学最具发展空间的领域之一.随着合成生物学的迅速发展,国际基因工程机器大赛(International Genetically Engineered Machine,iGEM)应运而生.iGEM竞赛项目基于合成生物学学科基础,应用现代生物学技术手段,立足解决社区和身边的实际生物...