转基因动物技术及其应用

转基因动物技术及其应用张涛（北京医科大学生物遗传教研室北京１０００８３）关键词转基因动物８０年代初期，由于分子生物学和动物实验胚胎学的发展和相互渗透，使科学家可以借助实验手段，将特定的目的基因从某一动物体分离出来，进行扩增或加工，再导人另一动物的早期...     

哺乳动物体细胞克隆及在动物转基因中的应用

哺乳动物体细胞克隆及转基因技术,近些年发展迅速。尤其1997年‘多利’羊的诞生,对推动哺乳动物克隆及转基因技术的发展具有里程碑式的意义。本文介绍了哺乳动物体细胞克隆的方法,存在的问题和体细胞克隆的机理研究。对这些方法和研究理论进行了讨论,并探讨如何利用克隆技术进行转基因动物的制备。     

转基因克隆动物技术及其应用

克隆动物是目前生物技术领域研究的热点之一,其科学意义和应用价值重大。根据现有理论和技术发展趋势,本文提出了转基因克隆动物的概念,即将转基因动物技术与克隆动物技术有机地结合,认为转基因克隆动物制作技术有望成为２１世纪创建遗传工程动物的主导性技术。     

转基因动物研究中存在问题

１５年来已建了许多用作研究人类疾病的转基因动物模型。转基因动物已作为研究代谢调节、分化、发育中有关基因功能的工具,它已作为一种分子农场可生产基因产品等。但要看到,转基因还有许多问题要解决。首先是转基因的阳性率低,这是受了许多因素如ＤＮＡ样品质量,分子大小和结构,动物种属,原核的可见性,注射针的调节和微注射技术等影响。通常转基因首建的阳性率在１０－２０％,有时甚至低于１％。在基因操作后,外源基因整合     

动物乳腺生物反应器与生物制药

利用动物乳腺生物反应器生产人类所需的药用蛋白,在制药领域展现出了广阔前景。综述了应用动物乳腺生物反应器制药的优越性和应用进展。     

电穿孔技术在转基因及动物克隆中的应用

电穿孔技术利用电场造成细胞膜的改变而将DNA导入细胞内,它还可用于细胞融合及动物克隆等。基因电转移的效率通常比化学法提高1—2个数量级,主要与脉冲波形、长度、缓冲液等有关。方波直流电脉冲应用广泛,在有关细胞核移植的多项研究报告中均指出它有重要作用。     

浅谈转基因克隆动物技术

根据现有理论和技术发展趋势,提出了转基因克隆动物的概念,即将克隆动物与转基因克隆动物技术有机地结合起来,认为转基因克隆动物制作 技术将有望成为下一世纪创建遗传工程动物的主导性技术。     

转基因克隆动物技术及其应用

克隆动物是目前生物技术领域研究的热点之一,其科学意义和应用价值重大。根据现有理论和技术发展趋势,本文提出了转基因克隆动物的概念,即将转基因动物技术与克隆动物技术有机地结合,认为转基因克隆动物制作技术有望成为21世纪创建遗传工程动物的主导性技术 。     

动物乳腺生物反应器的现状和趋势

利用转基因家畜的乳腺生产人类重组蛋白,可以高效获得安全、足量的药用蛋白。本文针对乳腺生物反应器的成功研制,从目的基因的选择、载体构建、转基因技术等方面探讨了动物乳腺生物反应器的研究现状。分析了提高转基因效率和外源蛋白表达水平的技术途径,提出了降低总体成本的战略措施。特别探讨了利用Cre-loxP系统发展“体细胞打靶体细胞核移植技术体系”,高效生产乳腺生物反应器动物的可能性。     

精子介导转基因动物的制备

采用直接注射的方法,将脂质体包裹的含人乳铁蛋白基因的重组质粒pLNCXHLF注入兔睾丸组织和羊输精管中,一个月后分别与正常雌兔及正常的母羊交配。所产仔兔均为活体,经PCR和Southern检测,转基因仔兔阳性率平均为35%(11/31);羔羊经引物F1/R1系统PCR检测阳性率为33.3％（4/12）,F2/R2系统PCR检测阳性率为25%（3/12）。将流产羔羊组织解剖后提取舌、肺、肝脏、肌肉、皮肤、脑、生殖腺、脾脏、小肠、心脏、肾脏共11种组织的总DNA进行PCR检测,发现：F1/R1和F2/R2系统PCR检测出阳性信号存在于不同组织;F2/R2 PCR系统检测阳性信号较F1/R1 PCR系统少;而且各种组织的外源DNA存在的拷贝数不等,造成阳性信号的强弱程度不同。结果表明：1）脂质体包裹外源基因转染精子的方法,可将外源基因导入受精卵,并得到了较高的转基因阳性率;2）精子携带外源DNA的整合过程是随机的,在受精过程和胚胎早期分化过程中可能发生了片段丢失、不完全整合或游离于基因组存在而产生嵌合体。本文的研究结果证明了该方法是一种简捷有效的新途径,为进一步深入探讨精子介导转基因动物制备可行性奠定了基础,给精子载体法制备转基因哺乳动物研究提供了有价值的参考。 Abstract:The recombinant plasmids pLNCXHLF fused with human lactoferritin gene were directly injected into male rabbit's testis and male goat's spermaductus.The transfected males were fertilized with females one month later.Using F1/R1 PCR system and southern blotting,the transgenic positive rate of rabbit offsprings genomic DNA was 35%(11/31).From the PCR results of F1/R1 and F2/R2 system,the transgenic positive rate of genomic DNA of goat offsprings were 33.3% (4/12)and 25%(3/12) respectively.We prepared genomic DNA from 11 kinds of tissues of goat offsprings,which were tongue,lung,liver,muscle,skin,brain,gonad,spleen,intestines,heart,and kidney.The result of F1/R1 PCR system indicated that the abilities to uptake exogenous DNA were various in different tissues;the positive signals of F2/R2 PCR system were feebler than the ones of F1/R1 PCR system,and the density of positive signals attributed to the amount of copies of exogenous DNA in the tissues.In this experiment,spermatozoa-mediated gene transfer can produce the transgenic animals after exogenous DNA being entrapped by liposome.But during the course of fertilization and the early process of embryo proliferation,the exogenous DNA had lost segments,partly integrated,or existed outside of genomic DNA.So the rate of chimera was relatively high.According to this result,this method is not only a simple and effective way to produce transgenic animals but also make references to other researchers to prepare transgenic mammals by this means.     

转基因动物的乳腺表达

转基因动物乳腺组织特异性表达异源基因是近年来基因工程中引人注目的途径.文章介绍了这一途径有关的乳汁蛋白基因、乳汁蛋白基因与异源基因的融合方式、重组基因的必要构成以及可能影响高效表达的因素.     

转基因动物在microRNA研究中的应用

MicroRNA是一类在转录后水平上调节基因表达的非编码小分子RNA,在生物体生理、病理等过程中发挥重要作用．MicroRNA功能的研究将是未来人们关注的焦点．通过转基因技术建立的多种动物模型在整体水平揭示了基因的功能．近年,以microRNA为研究对象的转基因动物模型数量不断增加,构建策略不断丰富．通过miRNA过表达、敲除及敲减等手段已揭示了miRNA在肿瘤、心血管系统疾病等多方面的作用．转基因动物正成为microRNA研究中不可或缺的工具．     

动物转基因高效表达策略研究进展

外源基因在动物体内、细胞内表达时,常由于各种原因导致基因沉默。目前,主要有两种策略用于打破该瓶颈:一是利用友好位点;二是优化外源基因顺式作用元件。利用友好位点就是通过定点整合技术将外源基因插入至活性转录区域;优化外源基因顺式作用元件即采用合适的启动子、增强子、内含子、染色质开放元件、核基质附着区等元件,构建出高效表达载体。对相关策略进行概述,为动物基因工程育种提供参考。     

转基因动物技术的研究进展及应用

文章综述了转基因动物的制作的主要方法及其优缺点,包括显微原核注射法、逆转录病毒感染法、胚胎干细胞介导法、体细胞核移植技术、精子载体法、胞浆内单精子注射法以及卵母细胞载体法等。阐述了转基因动物技术在人类疾病模型、生产人体器官、动物反应器和改良动物品种及其生产性能等方面的应用,并提出转基因动物技术存在的一些技术难题和安全性问题。     

转基因动物研究新进展

我们曾对转基因动物的制作方法、转基因动物研究的应用、转基因的表达特征及提高转基因表达的策略等作过专题综述＾「１」。而这之后在转基因动物的研究方面又获得了许多新进展,其中转线粒体动物的问世拓宽了转基因动物的研究内容。在制作转基因动物的方法上,１９９８－１９９９年世界上先后报道了三种新方法：即由Ｓｃｈｎｉｅｋ等报道的体细胞核移植技术实现转基因,由Ａｎｔｈｏｎｙ Ｗ．Ｓ．Ｃ等报道的通过用逆转录病毒载体感     

转基因动物乳腺生物反应器的发展概况及人工染色体在其中的应用

转基因动物乳腺生物反应器位点效应的影响是制备转基因动物乳腺生物反应器过程中的主要问题。酵母人工染色体（YAC）和细菌人工染色体（BAC）具有容量大的特性,可以将乳蛋白的整个基因座包括所有调控序列全部装载进去,有可能克服位点效应的影响,是一种理想的载体。YAC和BAC载体转基因技术可能成为避开基因打靶获得高效表达的转基因动物乳腺生物反应器的另一途径.     

Transgenic animal bioreactors

The production of recombinant proteins is one of the major successes of biotechnology. Animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animals are being used for this purpose. Milk, egg white, blood, urine, seminal plasma and silk worm cocoon from transgenic animals are candidates to be the source of recombinant proteins at an industrial scale. Although the first recombinant protein produced by transgenic animals is expected to be in the market in 2000, a certain number of technical problems remain to be solved before the various systems are optimized. Although the generation of transgenic farm animals has become recently easier mainly with the technique of animal cloning using transfected somatic cells as nuclear donor, this point remains a limitation as far as cost is concerned. Numerous experiments carried out for the last 15 years have shown that the expression of the transgene is predictable only to a limited extent. This is clearly due to the fact that the expression vectors are not constructed in an appropriate manner. This undoubtedly comes from the fact that all the signals contained in genes have not yet been identified. Gene constructions thus result sometime in poorly functional expression vectors. One possibility consists in using long genomic DNA fragments contained in YAC or BAC vectors. The other relies on the identification of the major important elements required to obtain a satisfactory transgene expression. These elements include essentially gene insulators, chromatin openers, matrix attached regions, enhancers and introns. A certain number of proteins having complex structures (formed by several subunits, being glycosylated, cleaved, carboxylated...) have been obtained at levels sufficient for an industrial exploitation. In other cases, the mammary cellular machinery seems insufficient to promote all the post-translational modifications. The addition of genes coding for enzymes involved in protein maturation has been envisaged and successfully performed in one case. Furin gene expressed specifically in the mammary gland proved to able to cleave native human protein C with good efficiency. In a certain number of cases, the recombinant proteins produced in milk have deleterious effects on the mammary gland function or in the animals themselves. This comes independently from ectopic expression of the transgenes and from the transfer of the recombinant proteins from milk to blood. One possibility to eliminate or reduce these side-effects may be to use systems inducible by an exogenous molecule such as tetracycline allowing the transgene to be expressed only during lactation and strictly in the mammary gland. The purification of recombinant proteins from milk is generally not particularly difficult. This may not be the case, however, when the endogenous proteins such as serum albumin or antibodies are abundantly present in milk. This problem may be still more crucial if proteins are produced in blood. Among the biological contaminants potentially present in the recombinant proteins prepared from transgenic animals, prions are certainly those raising the major concern. The selection of animals chosen to generate transgenics on one hand and the elimination of the potentially contaminated animals, thanks to recently defined quite sensitive tests may reduce the risk to an extremely low level. The available techniques to produce pharmaceutical proteins in milk can be used as well to optimize milk composition of farm animals, to add nutriceuticals in milk and potentially to reduce or even eliminate some mammary infectious diseases. This revised version was published online in August 2006 with corrections to the Cover Date.     

转基因植物检测体系在环境污染物突变性评价中的应用The

很多环境污染物都能对生物体产生诱变性,可利用多种测试材料与方法对其基因毒性进行检测。转基因植物检测体系不仅能对环境污染的诱变性大小进行评价,而且还可以进一步分析其突变的分子机理。本文介绍了转基因植株检测体系的应用前景。 Abstract: Environmental contaminants are powerful mutagenic factors for organisms. Several testing materials and methods have been used to assess the genotoxicity of environmental factors. Transgenic plants testing system can not only evaluate the level of genotoxicity, but also provide information on the genetic effects at molecular level. We introduce the use of transgenic plants in biomonitoring environmental factors.