RNAi在抗病毒防治领域的应用

RNAi是由双链RNA(dsRNA)所诱发的转录后水平上的基因沉默.由于对靶基因沉默作用的高度特异性和高效性,因此近年来用于肿瘤性疾病、感染性疾病、遗传性疾病等疾病的基因治疗研究,特别是在抗病毒领域的研究更是成为其应用热点之一.虽然目前RNAi已经较为广泛地应用于动物病毒及各种疾病病毒的基因治疗研究中,但其在应用过程中还有许多亟待解决的问题.本文就RNAi及其在抗病毒领域的应用研究和其存在的问题展开综述.     

类器官组织在感染性疾病研究中的应用

类器官(organoid)是体外3D培养组织干细胞所形成的多功能细胞团,具有自身增殖和多向分化的能力,在空间和结构上与来源器官组织的基因、结构和功能相似,可用于模拟体内组织细胞生长、分化及器官形成过程,在药物筛选与评价、生物医学材料及组织工程等方面具有重要的应用潜能。当前在感染性疾病研究方面,越来越多数据表明不同类器官组织可用于在体外模拟病原入侵和引发疾病过程。鉴于此,本文对类器官组织在病原感染相关疾病模型上的研究进展及其应用前景进行综述。     

共生病毒组的组成及其功能

共生病毒广泛参与机体各项生理和病理过程，近年来受到高度关注。共生病毒在自身免疫性疾病、变应性疾病、肿瘤发生、抗病毒免疫反应及炎症性疾病的发生、发展过程中发挥重要作用，其可通过调控免疫细胞的分化发育和功能行使，尤其是细胞因子的分泌进而参与其中。由于二代测序技术的发展，宏基因组学研究技术进展迅速，共生病毒与疾病进展的研究进一步加深了人们对共生病毒功能的理解。由于共生病毒在机体的分布、宿主遗传背景以及共生微生物的不同，共生病毒可能诱导机体产生多种有益或不利的免疫反应。本文简要阐述了共生病毒在机体的分布、组成和作用机制，及其在疾病诊断和治疗中的潜在意义，并对共生病毒未来在疾病诊疗中的潜在应用前景做一展望。     

非洲爪蟾在神经系统疾病研究中的应用

在生物医学研究领域,使用合适的动物模型研究大脑运行机制和疾病机理至关重要。作为经典的脊椎动物模型,非洲爪蟾在研究神经环路构建和功能以及神经疾病的分子机制中具有一定优势。因为非洲爪蟾的胚胎发育过程与人类器官形成过程相似,同时,神经系统疾病往往与神经系统功能异常和发育缺陷有关,所以一些人类疾病在基因和形态上的功能机制可以通过非洲爪蟾的胚胎发育模型进行在体研究,且神经前体细胞的增殖分化以及视觉刺激依赖的神经环路稳态和功能研究已经相对成熟。目前,该模型已经在癫痫和自闭症及表观遗传调控等相关疾病的研究上得到了应用。该文将分别介绍非洲爪蟾蝌蚪模型在环境毒物诱发疾病、神经发育障碍以及表观遗传疾病机制研究领域的最新进展。     

CRISPR/Cas9系统的发展及其在动物基因编辑中的应用 *

CRISPR/Cas9作为一种新型的基因编辑技术,主要在DNA水平对生物体的遗传信息进行修改,具有强大的基因编辑能力,目前已经被广泛应用于多个领域,包括基因功能研究、构建动物模型、家畜新品种的培育以及基因治疗等。CRISPR/Cas9技术的不断发展为生物学及医学领域的研究带来了革命性的突破,利用该技术构建基因突变小鼠有助于基因功能的研究,对于遗传疾病的治疗等具有重要的参考价值,同时还可以从基因组水平上有效改善家畜动物的生产性能,提高家畜动物的抗病能力。主要介绍了CRISPR/Cas系统的研究历程、结构与分类,详细阐述了CRISPR/ Cas9技术的作用机制及其在动物基因编辑中的应用,探讨了CRISPR/Cas9在基因编辑动物的制备中存在的问题及优化策略,并对其发展前景进行了展望。     

超级增强子研究进展

超级增强子是具有转录活性增强子的一个大簇,驱动控制细胞身份基因的表达,在发育和肿瘤等疾病发生过程中起到重要作用。和普通增强子相比,许多肿瘤细胞关键致癌基因是由超级增强子驱动,常见疾病如阿尔茨海默病等相关的变异显著富集于超级增强子。超级增强子在关键致癌基因的鉴定、疾病关联变异位点的发现等领域显示了巨大的应用潜力。本文首先概述了如何在全基因组水平进行增强子的鉴定,随后引入超级增强子的概念和鉴定方法,最后阐述了超级增强子的主要结构和功能特征,并对其在研究中的应用做了展望。     

基因编辑动物模型在人类疾病研究中的应用

近年来,随着以CRISPR/Cas9为代表的多种CRISPR系统的开发和不断改进,基因编辑技术逐渐完善,并广泛应用于人类疾病动物模型的制备。基因编辑动物模型为人类疾病的发病机理、病理过程以及预防和治疗等方面的研究提供了重要的素材。目前,用于人类疾病研究的基因编辑动物模型主要有小鼠、大鼠为代表的啮齿类动物模型和以猪为代表的大动物模型。其中啮齿类动物在机体各方面与人类差别较大,且寿命短,无法对人类疾病的研究和治疗提供有效评估和长期追踪;而猪在生理学、解剖学、营养学和遗传学等各方面与人类更接近,是器官移植和人类疾病研究领域重要的动物模型。文中主要介绍了基因编辑动物模型在神经退行性疾病、肥厚心肌病、癌症、免疫缺陷类疾病和代谢性疾病等5种人类疾病研究中的应用情况,以期为人类疾病研究及相关动物模型的制备提供参考。     

基质金属蛋白酶ADAMTS的研究进展

ADAMTS是含有TSP基序的分泌性多结构域蛋白水解酶。ADAMTS基因的突变或过表达与多种生理学和病理学过程相关。ADAMTS基因的过表达促进了细胞外基质成分的降解,加速了关节炎和动脉粥样硬化的疾病进程。ADAMTS基因的突变则与肿瘤的生长和侵袭以及遗传性发育紊乱等密切相关。该文将结合作者的研究工作,重点对ADAMTS-7的当前研究概况进行综述,讨论其结构、功能、调节及其在相关炎性疾病中的作用等。     

环状RNA翻译能力研究进展

随着高通量测序技术和翻译组学研究的快速发展,对环状RNA (circular RNA, circRNA)翻译能力的研究日益成为热点。已有研究表明,circRNA自身可以翻译为蛋白,其蛋白功能与人类疾病发生发展有着密切联系,而且其有望成为mRNA的理想替代品,未来可被广泛地应用在蛋白质工程。本文系统综述了circRNA来源、形成方式和主要特征、翻译蛋白的方式、翻译能力的鉴定和功能验证,归纳了近年来circRNA翻译在人类疾病中的研究进展及其在蛋白质工程的应用,并对后续研究关注的问题进行了展望,以期为相关领域的研究提供参考。     

miR-21在皮肤相关疾病及创伤愈合中的作用

microRNA-21(miR-21)是一种内源性非编码RNA,在细胞增殖、分化过程中发挥了重要的调控作用。近年来，miR-21作为广泛研究的miRNA,其在皮肤相关疾病及创伤愈合中的作用备受关注。研究表明，miR-21作为一个“广泛因子”,通过抑制不同靶基因(PTEN、TIMP、PDCD4等)的转录翻译过程，影响不同细胞(角质形成细胞、T细胞、纤维细胞等)的增殖、凋亡、迁移和侵袭。同时，还能通过不同信号通路，促进炎症的发生，在皮肤肿瘤、皮肤免疫性疾病、皮肤炎症性疾病、皮肤创伤及瘢痕组织形成中发挥重要作用。本文回顾了miR-21在不同的皮肤疾病(黑色素瘤、皮肤鳞状细胞癌、T细胞淋巴瘤、银屑病、硬皮病等)和创伤愈合中的参与机制，旨在深化对miR-21分子在皮肤相关疾病和创伤愈合中的认识，表明miR-21除了有作为皮肤性疾病诊断的生物标志物潜力和评价药物疗效的能力外，更有望成为一种新型的治疗手段，为临床治疗难题提供新方向。     

利用Red同源重组系统构建兔次黄嘌呤-鸟嘌呤磷酸核糖转移酶基因打靶载体

利用EL350基因工程菌进行同源重组,成功进行基因敲除已有报道,但利用该系统进行兔次黄嘌呤-鸟嘌呤磷酸核糖转移酶(Hypoxanthine guanine phosphoribosyl transferase,HPRT)基因突变和基因打靶方面的研究还没有报道。本实验首先在已经筛选到含有兔全长HPRT基因BAC克隆（LBNL1-304M19）的基础上,利用Red重组系统,通过Gap-Repair方式从此克隆上将一段47Kb无启动子的HPRT基因组片段（不含有第1个外显子）克隆到pBACLinkSp质粒上,产生pBACLinkSp-rHPRT质粒。然后基于pBACLinkSp-rHPRT质粒,设计不同的同源臂,从而删除了HPRT基因的不同编码区,成功构建了三个不同的HPRT基因打靶载体。同时对利用同源重组技术敲除不同大小的DNA片段的效率进行了研究。基于本实验所构建的三个不同的兔HPRT基因打靶载体,为探索兔成纤维细胞和胚胎干细胞基因打靶的适宜条件,及进一步获得兔HPRT基因敲除动物疾病模型奠定了基础。     

Effect of Genomic Location on Horizontal Transfer of a Recombinant Gene Cassette Between Pseudomonas Strains in the Rhizosphere and Spermosphere of Barley Seedlings

The use of genetically engineered bacteria in natural environments constitutes a risk of transfer of recombinant DNA to the indigenous bacteria. However, chromosomal genes are believed to be less likely to transfer than genes on mobilizable and conjugative plasmids. To study this assumption, horizontal transfer of a recombinant gene cassette inserted into the chromosome of a Pseudomonas stutzeri strain, into a mobilizable plasmid (pAGM42), and into a conjugative plasmid (pKJK5) isolated from barley rhizosphere was investigated. Horizontal transfer efficiencies of the gene cassette inserted into a conjugative plasmid was 8.20 × 10⁻³ transconjugants/(donors × recipients)^1/2 in the rhizosphere and 4.57 × 10⁻² transconjugants/(donors × recipients)^1/2 in the spermosphere. Mobilization of the plasmid pAGM42 by the plasmids RP4 and pKJK5 was also detected at high levels in the microcosms, transfer efficiencies were up to 4.36 × 10⁻³ transconjugants/(donors × recipients)^1/2. Transfer of chromosomal encoded genes could not be detected in the microcosms by conjugation or transformation. However, transformation did occur by using the same bacterial strains under laboratory conditions. The rhizosphere and especially the spermosphere thus proved to be hot spot environments providing favorable conditions for gene transfer by mobilization and conjugation, but these environments did not support transformation at a detectable level. Received: 21 July 2000 / Accepted: 21 August 2000     

Suicidal genetically engineered microorganisms for bioremediation: need and perspectives

In the past few decades, increased awareness of environmental pollution has led to the exploitation of microbial metabolic potential in the construction of several genetically engineered microorganisms (GEMs) for bioremediation purposes. At the same time, environmental concerns and regulatory constraints have limited the in situ application of GEMs, the ultimate objective behind their development. In order to address the anticipated risks due to the uncontrolled survival/dispersal of GEMs or recombinant plasmids into the environment, some attempts have been made to construct systems that would contain the released organisms. This article discusses the designing of safer genetically engineered organisms for environmental release with specific emphasis on the use of bacterial plasmid addiction systems to limit their survival thus minimizing the anticipated risk. We also conceptualize a novel strategy to construct "Suicidal Genetically Engineered Microorganisms (SGEMs)" by exploring/combining the knowledge of different plasmid addiction systems (such as antisense RNA-regulated plasmid addiction, proteic plasmid addiction etc.) and inducible degradative operons of bacteria.     

The construction and monitoring of genetically marked, plasmid-containing, naphthalene-degrading strains in soil

A genetically marked, plasmid-containing, naphthalene-degrading strain, Pseudomonas putida KT2442(pNF142::TnMod-OTc), has been constructed. The presence of the gfp gene (which codes for green fluorescent protein) and the kanamycin and rifampicin resistance genes in the chromosome of this strain allows the strain's fate in model soil systems to be monitored, whereas a minitransposon, built in naphthalene biodegradation plasmid pNF142, contains the tetracycline resistance gene and makes it possible to follow the horizontal transfer of this plasmid between various bacteria. Plasmid pNF142::TnMod-OTc is stable in strain P. putida KT2442 under nonselective conditions. The maximal specific growth rate of this strain on naphthalene was found to be higher than that of the natural host of plasmid pNF142. When introduced into a model soil system, the genetically marked strain is stable and competitive for 40 days. The transfer of marked plasmid pNF142::TnMod-OTc to natural soil bacteria, predominantly fluorescent pseudomonads, has been detected.     

Dynamics of Horizontal Gene Transfer and the Ecological Risk Assessment of Genetically Engineered Organisms

The extensive published discussion of potential ecological impacts of introduced genetic sequences and genetically engineered organisms has lacked a quantified delineation of the critical questions for the estimation of risk. Ultimately, the ecological risk assessment of introduced gene sequences is the application of evolution, population genetics, and ecology to risk estimation and decision making. This paper provides a framework for the estimation of risk due to introduced sequences in bacteria, and the principles should also hold for many diploid species. Horizontal genetic exchange poses new challenges for ecological risk assessment. Plasmid transfer can occur without any impacts, although the sequence can become ubiquitous in the population. Conversely, the introduction of a plasmid can change the dynamics of the host population, potentially altering the population minimum and maximum characteristics of its dynamics. Because of genetic exchange, new genetic information is unlikely to be constrained among one type of prokaryote. An example of the use of the model is given using genetic exchange data from a series of published soil microcosm experiments. The model demonstrates the increase in plasmid frequency when using experimentally derived conjugation frequencies. Application of these results to ongoing discussion of the risks of genetically engineered organisms is presented. Particular attention is paid to the transfer of genetic material and the resultant changes in host population dynamics.     

Gene transfer between Salmonella enterica serovar Typhimurium inside epithelial cells

Virulence and antibiotic resistance genes transfer between bacteria by bacterial conjugation. Conjugation also mediates gene transfer from bacteria to eukaryotic organisms, including yeast and human cells. Predicting when and where genes transfer by conjugation could enhance our understanding of the risks involved in the release of genetically modified organisms, including those being developed for use as vaccines. We report here that Salmonella enterica serovar Typhimurium conjugated inside cultured human cells. The DNA transfer from donor to recipient bacteria was proportional to the probability that the two types of bacteria occupied the same cell, which was dependent on viable and invasive bacteria and on plasmid tra genes. Based on the high frequencies of gene transfer between bacteria inside human cells, we suggest that such gene transfers occur in situ. The implications of gene transfer between bacteria inside human cells, particularly in the context of antibiotic resistance, are discussed.     

An efficient CRISPR/Cas9-based genome editing system for alkaliphilic Bacillus sp. N16-5 and application in engineering xylose utilization for D-lactic acid production

Alkaliphiles are considered more suitable chassis than traditional neutrophiles due to their excellent resistance to microbial contamination. Alkaliphilic Bacillus sp. N16-5, an industrially interesting strain with great potential for the production of lactic acid and alkaline polysaccharide hydrolases, can only be engineered genetically by the laborious and time-consuming homologous recombination. In this study, we reported the successful development of a CRISPR/Cas9-based genome editing system with high efficiency for single-gene deletion, large gene fragment deletion and exogenous DNA chromosomal insertion. Moreover, based on a catalytically dead variant of Cas9 (dCas9), we also developed a CRISPRi system to efficiently regulate gene expression. Finally, this efficient genome editing system was successfully applied to engineer the xylose metabolic pathway for the efficient bioproduction of D -lactic acid. Compared with the wild-type Bacillus sp. N16-5, the final engineered strain with XylR deletion and AraE overexpression achieved 34.3% and 27.7% increases in xylose consumption and D -lactic acid production respectively. To our knowledge, this is the first report on the development and application of CRISPR/Cas9-based genome editing system in alkaliphilic Bacillus, and this study will significantly facilitate functional genomic studies and genome manipulation in alkaliphilic Bacillus, laying a foundation for the development of more robust microbial chassis.     

Leaching of Pseudomonas aeruginosa, and transconjugants containing pR68.45 through unsaturated, intact soil columns

Abstract Leaching of genetically engineered microbes (GEMs) through soil is a significant concern related to groundwater quality. The objective of this study was to examine the leaching, survival and gene transfer of a genetically engineered microbe and indigenous recipients of pR68.45 in nonsterile, undisturbed soil columns. Pseudomonas aeruginosa PAO25, containing the plasmid R68.45, was added to the surface of undisturbed soil columns (10 cm diameter × 80 cm length). Unsaturated flow conditions were maintained by 100 ml daily additions of 2 mM CaCl₂ for a period of 70 days. The population of the GEM exhibited a significant ( P = 0.05) linear decline with time. The GEM leached only to a depth of 30–40 cm in 70 days. Transfer of pR68.45 was shown to occur from P. aeruginosa into the indigenous bacterial population although relatively low numbers of transconjugants were observed (log 2 cfu g⁻¹ dry soil). The number of transconjugants also decreased with depth and time. Leaching of transconjugants, however, occured more readily than that of the GEM, probably as a result of plasmid transfer into smaller, more mobile bacteria. At 70 days incubation, no GEMs were detected in the columns, while transconjugants were observed at several depths. These results demonstrate the importance of examining both the survival and movement of GEMs and transconjugants in soil.     

Mechanisms of, and barriers to, horizontal gene transfer between bacteria

Bacteria evolve rapidly not only by mutation and rapid multiplication, but also by transfer of DNA, which can result in strains with beneficial mutations from more than one parent. Transformation involves the release of naked DNA followed by uptake and recombination. Homologous recombination and DNA-repair processes normally limit this to DNA from similar bacteria. However, if a gene moves onto a broad-host-range plasmid it might be able to spread without the need for recombination. There are barriers to both these processes but they reduce, rather than prevent, gene acquisition.     

The Construction and Monitoring of Genetically Tagged,Plasmid-Containing,Naphthalene-Degrading Strains in Soil

A genetically tagged, plasmid-containing, naphthalene-degrading strain, Pseudomonas putida KT2442(pNF142:: TnMod-OTc), has been constructed. The presence of the gfp gene (which codes for green fluorescent protein) and the kanamycin and rifampicin resistance genes in the chromosome of this strain allows the strain’s fate in model soil systems to be monitored, whereas a minitransposon, inserted into naphthalene biodegradation plasmid pNF142 and containing the tetracycline resistance gene, makes it possible to follow the horizontal transfer of this plasmid between various bacteria. Plasmid pNF142::TnMod-OTc is stable in strain P. putida KT2442 under nonselective conditions. The maximal specific growth rate of this strain on naphthalene is found to be higher than that of the natural host of plasmid pNF142. When introduced into a model soil system, the genetically tagged strain is stable and competitive for 40 days. The transfer of labeled plasmid pNF142::TnMod-OTc to natural soil bacteria, predominantly fluorescent pseudomonads, has been detected.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 526–532.Original Russian Text Copyright © 2005 by Filonov, Akhmetov, Puntus, Esikova, Gafarov, Izmalkova, Sokolov, Kosheleva, Boronin.