转基因小鼠中应用RNAi的初步探讨

RNA干扰(RNA interference,RNAi)是通过双链RNA介导,特异性地降解相应序列的mRNA,从而导致转录后水平的基因沉默。近年来,利用小分子RNA(small interfering RNA,siRNA)介导的RNAi技术已越来越多的应用于哺乳动物细胞中。同时,在活体中利用RNAi研究多种生物基因功能也成为一种有效的手段。文章旨在介绍在转基因小鼠中RNAi的应用。     

哺乳动物转基因载体——piggyBac转座子

piggyBac转座子具有识别位点特异性5'-TTAA-3',剪切和插入都不留下印迹,且高效转座。基于piggyBac转座子的特性,借助载体系统,已应用于哺乳动物转基因的研究。此外,piggyBac转座子还应用于基因诱导突变和基因治疗等领域。RNAi技术作为基因功能研究的一个工具,在应用于克服畜牧业中家畜的生产繁殖障碍等方面已取得一定研究成果。本文对piggyBac转座子的结构和特性、转座机制及影响因素和在动物中应用等方面进行总结,为RNAi技术借助piggyBac转座子用于哺乳动物转基因提供理论依据。     

转基因猪技术及其在农业上的应用

转基因猪技术不仅可用于猪的育种,还可应用于生物医药领域。该技术对养猪业生产水平的提高和人类健康的改善均有重要的应用价值。本文从转基因猪制备所需的显微操作技术和介导外源基因整合方法两方面对转基因猪技术的研究进展进行综述,并简要概括转基因猪技术在农业领域的应用现状。     

哺乳动物RNAi抗病毒机制研究进展

RNAi是由双链小RNA(siRNA)介导的以序列特异性方式诱导同源mRNA降解的一种新技术,由于其特异性和高效性,已成为当前广泛应用的特异性基因阻断技术。虽然内源性RNAi机制在果蝇、秀丽隐杆线虫、拟南芥等低等动植物中的抗病毒侵染作用已较为明确,但在高等动物尤其在哺乳动物抗病毒感染中的研究一直处于探讨阶段。近期,《Science》等顶级学术期刊上,连续发表将RNAi应用于哺乳动物病毒性疾病的治疗性研究。发现去除病毒中的抑制蛋白,乳鼠体内产生大量内源性siRNA并对病毒进行攻击,且成功抑制病毒扩增,使得乳鼠存活。研究结果均证明RNAi在哺乳动物中也具有抗病毒功能,从而为高等动物病毒病的预防及治疗开辟了一条新途径。本文简单回顾RNAi的抗病毒作用机制,叙述RNAi在哺乳动物水平上的研究情况,尤其是近期应用RNAi在小鼠以抵抗病毒对机体的感染和相关应用与安全性问题取得的新进展。     

精子介导法制备GHRH转基因猪的研究

以eGFP为报告基因,生长激素释放激素(GHRH)为目的基因,利用精子介导的基因转移方法,将构建慢病毒载体质粒LV-EGFP-GHRH直接和猪精子孵育,再通过体外人工受精建立GHRH转基因猪模型.经PCR检 测,52头后代中发现7头阳性个体,转基因效率为7.42%.本研究探索了应用精子介导的转基因方法将GHRH转移到动物体内,为猪的转基因育种提供了新的依据.     

RNA干扰技术在动物抗病育种中的应用前景

动物疾病尤其是病毒性疾病一直是畜牧业亟待解决的问题,随着转基因技术的出现,生产遗传修饰动物来抵抗特定传染病的策略备受关注。从长远利益来看,这种基因工程动物从遗传本质上提高了畜禽的抗病能力。转基因抗病育种在未来可能成为减少动物传染性疾病的最有前途的方法之一。RNA干扰是双链RNA特异性诱导同源基因表达沉默的现象。研究证实,RNA干扰在细胞水平,小鼠模型以及动物个体的抗病毒效果是非常明显的,RNA干扰技术作为抗病毒策略为生产抗病转基因动物提供了光明的应用前景。RNA干扰技术介导的抗病转基因动物的研究相继取得了阶段性进展,抗疯牛病转基因羊和牛以及抗内源性逆转录病毒猪已经成功获得,虽然目前获得的转基因动物类型有限,但为继续生产抗病转基因家畜提供了有效的参考和依据。     

ie-1和lef-1基因dsRNA表达元件转染及转化细胞对家蚕核型多角体病毒增殖的抑制

通过RNAi介导可以抑制病毒增殖,将相应的RNAi元件通过转基因转入宿主有可能使宿主对病毒产生一定的抗性。根据家蚕核型多角体病毒(BmNPV)基因组中的病毒复制必需基因ie-1、lef-1设计相应的RNA干扰区段,并构建相应的转基因载体转入家蚕BmN细胞中,瞬时表达结果显示转染了转基因RNAi载体的BmN细胞均对BmNPV有一定的抑制作用。IE dsRNA在病毒滴度为10-4时有抑制作用,在病毒滴度为10-5时有比较好的作用;LEF dsRNA则在病毒滴度为10-5时有抑制作用,在病毒滴度为10-6时有较好的抑制作用。通过转基因及G418筛选后,转基因IE dsRNA细胞在病毒滴度为10-4显示出一定的抑制作用,在病毒滴度为10-5表现了明显的抑制作用;而转基因LEF dsRNA细胞在病毒滴度为10-5有一定的抑制作用,但只维持了一个时段。     

世界首例ROSA26基因敲入猪模型诞生

正近日,中科院广州生物医药与健康院研究员赖良学与美国密西根大学教授王忠合作,获得了世界首例ROSA26定点基因敲入猪模型。据称,该研究成功地实现了猪重组酶介导的基因交换,从而解决了一直困扰转基因猪研究领域效率低下、表型不确定的问题。转基因猪在农业新品种培育和生物医药研究中具有非常重要的应用价值。长期以来,由于缺乏猪胚胎干细胞,转基因猪的制备主要依赖体细胞基因     

哺乳动物中RNA干扰研究进展

RNA干扰(RNAi)由双链RNA引起,广泛存在于动、植物中的序列特异性转录后基因沉默,是生物体在进化过程中,抵御病毒感染及由于重复序列和突变引起的基因组不稳定性的保护机制。论文从RNAi现象、RNAi发生机制、哺乳动物中的RNAi现象和RNAi与抗病毒感染等几个方面做了综述,详细介绍RNAi在哺乳动物中的应用。     

RNA干扰及其抗口蹄疫病毒复制的研究进展

RNA干扰（RNA interference,RNAi）是指由双链RNA（double strand RNA,dsRNA）介导的序列特异性RNA降解作用。已经证明,在植物和昆虫细胞中RNAi是其主要的抗病毒机制,但迄今为止,几乎没有发现哺乳动物细胞感染病毒后能自发诱导有效的抗病毒RNAi反应。为此,通过人工方法在哺乳动物细胞中建立有效的抗病毒RNAi便成了国内外学者孜孜探索的重要抗病毒策略之一。目前,RNAi的分子机制及其功能仍然有待更加深入地研究与阐明,但它作为一种反向遗传学手段已经在基因组结构与功能研究中得到广泛运用,不仅在抗多种哺乳动物病毒的研究方面取得了令人振奋的结果,而且已作为一种治疗策略运用于人类抵抗重大遗传性和病毒性疾病的研究当中。笔者对RNAi的分子机制,及其抗口蹄疫病毒复制的相关研究进展作了有关介绍。     

A transgenic Marc-145 cell line of piggyBac transposon-derived targeting shRNA interference against porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome (PRRS) is now considered to be one of the most important diseases in countries with intensive swine industries. The two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. In this study, we designed 5 of the small hairpin RNAs (shRNAs) targeting the GP5 and M gene of PRRSV respectively, and investigated their inhibition to the production of PRRSV. The highest activity displayed in shRNAs of the ORF6e sequence (nts 261-279), which the inhibition rate reached was 99.09%. The result suggests that RNAi technology might serve as a potential molecular strategy for PRRSV therapy. Furthermore, the transgenic Marc-145 cell line of piggyBac transposon-derived targeting shRNA interference against PRRS virus was established. It presented stable inhibition to the replication and amplification of PRRS. The work implied that shRNAs targeting the GP5 and M gene of PRRSV may be used as potential RNA vaccines in vivo, and supplied the screening methods of transformed pig embryonic fibroblast which are prerequisite for the disease-resistant transgenic pigs to PRRS.     

Successful suppression of endogenous α-1,3-galactosyltransferase expression by RNA interference in pig embryos generated in vitro

RNA interference (RNAi) technology using small interfering RNAs (siRNA) has been widely used as a powerful tool to knock down gene expression in various organisms. In pig preimplantation embryos, no attempt to suppress the target gene expression with such technology has been made. The purpose of this study is to demonstrate that the RNAi technology is useful for suppression of endogenous target gene expression at an early stage of development in pigs. Alpha-1,3-Galactosyltransferase (α-GalT) is an enzyme that creates the Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species, and removal of the epitope is considered to be a prerequisite for pig-to-human xenotransplantation. We decided to suppress the endogenous α-GalT mRNA expression in pig early embryos, since reduction of α-GalT synthesis is easily monitored by cytochemical staining with Bandeiraea simplicifolia isolectin-B(4), a lectin that specifically binds to the α-Gal epitope, and by RT-PCR analysis. Cytoplasmic microinjection of double-stranded RNA and pronuclear injection of an siRNA expression vector into the embryos generated in vitro resulted in a significant reduction in expression of the α-GalT gene and α-Gal epitope in blastocysts, at which stage the α-Gal epitope is abundantly expressed. Somatic cell nuclear transfer of embryonic fibroblasts stably transfected with an siRNA expression vector also led to a significant reduction in the level of α-GalT mRNA synthesis together with decreased amounts of the α-Gal epitope at the blastocyst stage. These results indicate that the RNAi technology is useful for efficient suppression of a target gene expression during embryogenesis in pigs and suggest the possibility of production of siRNA-expressing pigs for use in xenotransplantation.     

诱导多能干细胞技术在猪转基因研究中的应用前景

长期以来,猪都是科学研究中重要的生物模型,转基因猪在生物模型、器官移植和动物良种繁育等方面有着极其重要的研究及应用价值,而现在的转基因克隆猪的成功率及成活率都普遍偏低。诱导多能干细胞技术在再生医学和畜牧业等领域有着广泛而诱人的应用前景,但目前还较少被应用于转基因猪的研究中。作者对转基因技术和诱导多能干细胞技术的发展历程,特别是诱导多能干细胞技术在猪转基因研究中的应用进行简述,以期为开展相关研究的科研人员提供一定的参考。     

CRISPR/Cas9技术在猪、鸡中的应用研究进展

基因编辑是利用核酸酶在基因组的特定位点产生DNA双链断裂，从而触发细胞自身的DNA损伤修复机制，实现对靶基因序列进行位点特异性修饰。规律成簇间隔短回文重复序列（clustered regularly interspersed short palindromic repeat，CRISPR）及其相关蛋白9（Cas9）系统作为第3代基因编辑技术在农业和基因治疗研究中发挥着重要作用，与传统的锌指核酶和转录激活因子样效应物核酶基因编辑方法相比，它可以靶向基因的任何位点引起DNA双链断裂，实现目标基因的精准敲除或插入外源片段，并能快速、高效地修饰基因组，包括基因敲除、敲入、抑制、激活等，是应用最广泛的基因编辑工具。CRISPR/Cas9技术的出现彻底改变了生命科学、医学和遗传学研究现状。近年来，利用CRISPR/Cas9技术对动物基因组进行修饰，开启了畜禽分子育种的新纪元，不仅极大地推动了现代畜禽养殖技术的发展，而且为人类医学研究做出了特殊贡献，特别是在猪和鸡上。作者以猪和鸡为对象，综述了CRISPR/Cas9技术在异体器官移植供体、人类疾病的生物模型、抗病育种材料、全基因组高通量筛选等方面的研究进展，以及如何利用CRISPR技术快速又简单地生产出基因编辑猪、鸡，为推动CRISPR技术制备其他基因编辑动物提供参考依据。     

Transgenic pigs for xenotransplantation for humans]

Transgenic livestock have been generated via microinjection of DNA-constructs into pronuclei of zygotes. However, efficiency is low and only 1-3% transgenic offspring are to be obtained. Integration of the transgene occurs at random and expression is independent from the number of integrated copies but can be affected by the integration site. To overcome the shortage of human organs, transgenic pigs have been generated that express human complement regulatory genes. This approach enables to overcome the hyperacute rejection response as shown by an average survival rate (40-90 days) of the immunosuppressed primate recipients receiving a heart from a transgenic pig. It is expected that transgenic pigs would be available as organ donors in the next 5-10 years. A major prerequisite, however, is the prevention of the potential transfer of pathogenic microorganisms, in particular porcine endogenous retroviruses (PERV). Improvements of the efficiency in the generation of transgenic pigs will be achieved by the use of genetically modified donor cells in nuclear transfer technology (cloning).     

The plasmid constructs producing shRNA corresponding to the conserved 3D polymerase of Foot and Mouth Disease virus protects guinea pigs against challenge virus

RNA interference (RNAi) has been used as an effective antiviral strategy for its specific silencing of viral gene expression in mammalian cells. In this study, shRNA targeting two regions of Foot and Mouth Disease Virus (FMDV) i.e. 3D and 5'UTR which are very essential in virus replication were evaluated. The constructs were made using h7K RNA polymerase III promoter. We investigated in vivo inhibitory effect of shRNA on FMDV replication in BHK-21 cells and guinea pigs. The results showed that transfection of 3D shRNA could reduce virus growth by three folds when cells were challenged with 10(2) TCID(50) of FMDV. Pretreated guinea pigs with 3DshRNA were protected 80% with 10(3) GPID(50) of FMDV. As a first report in guinea pigs which are recognized animal model for FMD vaccine potency testing, the study suggests that shRNA could be a viable therapeutic approach to control severity of FMD infection and spread.     

Transgenic mammalian species, generated by somatic cell cloning, in biomedicine, biopharmaceutical industry and human nutrition/dietetics--recent achievements

Somatic cell cloning technology in mammals promotes the multiplication of productively-valuable genetically engineered individuals, and consequently allows also for standardization of transgenic farm animal-derived products, which, in the context of market requirements, will have growing significance. Gene farming is one of the most promising areas in modern biotechnology. The use of live bioreactors for the expression of human genes in the lactating mammary gland of transgenic animals seems to be the most cost-effective method for the production/processing of valuable recombinant therapeutic proteins. Among the transgenic farm livestock species used so far, cattle, goats, sheep, pigs and rabbits are useful candidates for the expression of tens to hundreds of grams of genetically-engineered proteins or xenogeneic biopreparations in the milk. At the beginning of the new millennium, a revolution in the treatment of disease is taking shape due to the emergence of new therapies based on recombinant human proteins. The ever-growing demand for such pharmaceutical or nutriceutical proteins is an important driving force for the development of safe and large-scale production platforms. The aim of this paper is to present an overall survey of the state of the art in investigations which provide the current knowledge for deciphering the possibilities of practical application of the transgenic mammalian species generated by somatic cell cloning in biomedicine, the biopharmaceutical industry, human nutrition/dietetics and agriculture.     

表达短ie-1 dsRNA的转化细胞对家蚕核型多角体病毒的抑制作用

为了利用RNAi技术提高家蚕对核型多角体病毒(BmNPV)的抗性,根据BmNPV复制必需基因ie-1设计其相应的dsRNA,构建带有ie-1 dsRNA表达盒的转基因载体piggyantiIE-Neo,结果显示:表达短ie-1 dsRNA的稳定转化Bm细胞,对BmNPV的增殖表现出抑制作用,但在病毒感染后期,由于病毒恢复增殖导致RNAi的效果被掩盖。通过反向PCR分析外源DNA片段插入基因组位点,结果显示:在转化细胞中,外源DNA可通过随机整合或按照piggyBac特定的转座位点TTAA插入细胞基因组。