基于核酸侵入反应的基因突变检测方法研究进展

肿瘤靶向药物治疗需要检测特异性的基因突变。尽管已开发出多种基于模板扩增的基因突变检测方法,但由于存在扩增产物交叉污 染的风险,其应用受到极大限制。基于信号扩增的核酸侵入反应,由于不存在扩增产物污染的风险,并且具有良好的单碱基识别特异性, 非常适用于基因突变的检测。因此,一系列基于核酸侵入反应的基因突变检测方法应运而生。综述若干基于核酸侵入反应的基因突变检测 方法原理与应用研究。     

Advances in <Emphasis Type="Italic">Drosophila</Emphasis> gene targeting and related techniques

Functional biological research has benefited tremendously by analyses of the phenotypes of mutant organisms which can be generated through targeted mutation of genes. In Drosophila, compared with random mutagenesis methods gene targeting has gained its popularity because it can introduce any desired mutation into a gene of interest. However, applications of gene targeting have been limited because the targeting efficiency varies with different genes, and the time and labor of targeting procedure are intensive. Nevertheless, improvement of gene targeting and development of its variant technologies have received much attention of scientists. Here we review recent progress that has been made in expanding the applications of gene targeting, which include the ΦC31 integration system and zinc-finger nucleases induced gene targeting, and new strategies that generate more efficient and reliable gene targeting.     

水稻大粒种质资源和遗传分析

谷粒重量是构成产量的三要素之一,对提高水稻产量具有重要意义.本文概述了国内外水稻大粒种质资源的现状,同时对粒重基因遗传分析的研究进展进行了综述.粒重是一个受多基因控制的数量性状,目前定位的粒重数量性状位点至少达89个、精细定位1个粒重基因gw3.1和1个长粒基因Lk-4（t）以及克隆1个粒重基因GS3,并在此基础上讨论了粒重在育种上的应用.     

水稻大粒种质资源和遗传分析

谷粒重量是构成产量的三要素之一, 对提高水稻产量具有重要意义。本文概述了国内外水稻大粒种质资源的现状, 同时对粒重基因遗传分析的研究进展进行了综述。粒重是一个受多基因控制的数量性状, 目前定位的粒重数量性状位点至少达89个、精细定位1个粒重基因gw3.1和１个长粒基因Lk-4(t)以及克隆1个粒重基因GS3, 并在此基础上讨论了粒重在育种上的应用。     

植物基因转化技术在苹果遗传改良中的应用

近年来,苹果基因转化技术研究取得了较大进展,本文从转化体系,选择标记,报告基因及目的基因的遗传学行为等方面综述了苹果遗传转化研究发展现状,着重论述了其在苹果遗传改良中的应用,并对植物基因转化技术在莱果上的应用前景和应注意的几个问题进行了讨论。     

Applications of EcR gene switch technology in functional genomics

Genetic engineering of plants using transgenic technology is targeted to enhance agronomic performance or improved quality traits in a wide variety of plant species, and has become a fundamental tool for basic research in plant biotechnology. Constitutive promoters are presently the primary means used to express transgenes in plants. However, inducible gene regulation systems based on specific chemicals have many potential applications in agriculture and for enhancing the basic understanding of gene function. As a result, several gene switches have been developed. The ecdysone receptor gene switch is one of the best inducible gene regulation systems available, because the chemical, methoxyfenozide, required for its regulation is registered for field use. An EcR gene switch with a potential for use in large-scale field applications has been developed by adopting a two-hybrid format. In a two-hybrid switch format, the GAL4 DNA binding domain (GAL4 DBD) was fused to the ligand binding domain (LBD) of the Choristoneura fumiferana ecdysone receptor (CfEcR); and, the VP16 activation domain (VP16 AD) was fused to the LBD of Locust migratoria retinoid X receptor (LmRXR). The sensitivity of the CfEcR gene switch was improved from micromolar to nanomolar concentrations of ligand by using the CfEcR:LmRXR two-hybrid switch. In this report, we demonstrate the utility of CfEcR:LmRXR two-hybrid gene switch in functional genomics applications for regulating the expression of a Superman-like single zinc finger protein 11 (ZFP11) gene in both Arabidopsis and tobacco transgenic plants.     

Toward predictive engineering of gene circuits

Many synthetic biology applications rely on programming living cells using gene circuits – the assembly and wiring of genetic elements to control cellular behaviors. Extensive progress has been made in constructing gene circuits with diverse functions and applications. For many circuit functions, however, it remains challenging to ensure that the circuits operate in a predictable manner. Although the notion of predictability may appear intuitive, close inspection suggests that it is not always clear what constitutes predictability. We dissect this concept and how it can be confounded by the complexity of a circuit, the complexity of the context, and the interplay between the two. We discuss circuit engineering strategies, in both computation and experiment, that have been used to improve the predictability of gene circuits.     

Non-polymerase-cycling-assembly-based chemical gene synthesis: strategies, methods, and progress

Chemical gene synthesis is a powerful tool for basic biological research and biotechnology applications. During the last 30 years, major advances have been made in the chemical synthesis of DNA sequences ranging from fragments of <1 kb to multi-gene sequences of >30 kb. There is a need for simple, reproducible, less error-prone and cost-effective methods that guarantee successful synthesis of the desired genes and are amenable to automation. Many polymerase chain reaction (PCR)-based and non-polymerase-cycling-assembly (PCA)-based strategies have been developed for chemical gene synthesis. The PCR-based method has been the subject of several recent reviews. Here, we provide an overview of the progress in non-PCA-based chemical gene synthesis using different strategies and methods, including enzymatic gene synthesis, annealing and ligation reaction, simultaneous synthesis of two genes via a hybrid gene, shotgun ligation and co-ligation, insertion gene synthesis, gene synthesis via one strand of DNA, template-directed ligation, ligase chain reaction, microarray-mediated gene synthesis, Blue Heron solid support technology and Sloning building block technology. The fundamental principle underlying each strategy, an example where applicable, and the advantages and disadvantages are discussed. The emphasis is on discussion of the most recent technologies and their potential applications, particularly for microarray-based genomics research.     

重组腺相关病毒规模化生物包装技术

重组腺相关病毒(Recombinant adeno-associated virus,rAAV)的诸多优点使其成为具有巨大潜力的人类基因治疗载体。人类基因治疗临床前和临床研究以及基因治疗产品的市场化要求rAAV载体生产的规模化。自1989年野生型的腺相关病毒序列被Samulski报道以来,rAAV的生产工艺已经从传统的质粒共转染发展到应用包装细胞系和生产细胞系,包装细胞也从"人体细胞"衍化到"昆虫细胞"。目前rAAV的生产规模和病毒载体质量都完全可以符合临床应用要求,有效地促进rAAV在基因治疗临床上的广泛运用。以下将着重介绍rAAV规模化生物包装技术的发展趋势,尤其各种规模化生产系统的要点。     

Foxtail mosaic virus: A tool for gene function analysis in maize and other monocots

Many plant viruses have been engineered into vectors for use in functional genomics studies, expression of heterologous proteins, and, most recently, gene editing applications. The use of viral vectors overcomes bottlenecks associated with mutagenesis and transgenesis approaches often implemented for analysis of gene function. There are several engineered viruses that are demonstrated or suggested to be useful in maize through proof-of-concept studies. However, foxtail mosaic virus (FoMV), which has a relatively broad host range, is emerging as a particularly useful virus for gene function studies in maize and other monocot crop or weed species. A few clones of FoMV have been independently engineered, and they have different features and capabilities for virus-induced gene silencing (VIGS) and virus-mediated overexpression (VOX) of proteins. In addition, FoMV can be used to deliver functional guide RNAs in maize and other plants expressing the Cas9 protein, demonstrating its potential utility in virus-induced gene editing applications. There is a growing number of studies in which FoMV vectors are being applied for VIGS or VOX in maize and the vast majority of these are related to maize–microbe interactions. In this review, we highlight the biology and engineering of FoMV as well as its applications in maize–microbe interactions and more broadly in the context of the monocot functional genomics toolbox.     

Lentiviral Vector-Mediated Gene Transfer and RNA Silencing Technology in Neuronal Dysfunctions

Lentiviral-mediated gene transfer in vivo or in cultured mammalian neurons can be used to address a wide variety of biological questions, to design animals models for specific neurodegenerative pathologies, or to test potential therapeutic approaches in a variety of brain disorders. Lentiviruses can infect non-dividing cells, thereby allowing stable gene transfer in post-mitotic cells such as mature neurons. An important contribution has been the use of inducible vectors: the same animal can thus be used repeatedly in the doxycycline-on or -off state, providing a powerful mean for assessing the function of a gene candidate in a disorder within a specific neuronal circuit. Furthermore, lentivirus vectors provide a unique tool to integrate siRNA expression constructs with the aim to locally knockdown expression of a specific gene, enabling to assess the function of a gene in a very specific neuronal pathway. Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in the brain. Therefore, the use of lentiviruses for stable expression of siRNA in brain is a powerful aid to probe gene functions in vivo and for gene therapy of diseases of the central nervous system. In this chapter I review the applications of lentivirus-mediated gene transfer in the investigation of specific gene candidates involved in major brain disorders and neurodegenerative processes. Major applications have been in polyglutamine disorders, such as synucleinopathies and Parkinson’s disease, or in investigating gene function in Huntington’s disease, dystonia, or muscular dystrophy. Recently, lentivirus gene transfer has been an invaluable tool for evaluation of gene function in behavioral disorders such as drug addiction and attention-deficit hyperactivity disorder or in learning and cognition.     

In pursuit of new developments for gene therapy of human diseases

Gene therapy aims at transferring a therapeutic gene into human somatic cells in order to treat a disease. Originally addressed to hereditary genetic disorders, gene therapy has found therapeutic applications in cancer, infectious diseases and degenerative disorders, particularly those of the nervous system. Although gene transfer into humans has been demonstrated in several clinical trials, with more than 300 currently underway worldwide, there is still no single outcome that undoubtedly showed a consistent benefit for the patient. Nevertheless, the expectations for gene therapy are still high, and the prospects of future clinical success are increasing together with the growing of the field. The development of better delivery systems specifically tailored to individual diseases, with sustained expression of the therapeutic gene in the appropriate cells, will in the end make possible true therapeutic applications of human gene transfer.     

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.     

DNA-damaging agents greatly increase the transduction of nondividing cells by adeno-associated virus vectors.

None of the vector systems currently available for gene therapy applications have been shown to be capable of both efficient gene transfer into nondividing cells and long-term expression through stable integration into host cell DNA. While integrating vectors based on adeno-associated virus are capable of mediating gene transfer into nondividing cells, this process is 200-fold less efficient than transduction of dividing cells. We demonstrate that the transduction efficiency of adeno-associated virus vectors can be increased by treatment with DNA-damaging agents. Nondividing cells are especially responsive, with increases in transduction efficiency of up to 750-fold. This finding has the potential to facilitate gene therapy applications requiring gene transfer to nondividing cells.     

Factors affecting the genetic engineering of plants by microprojectile bombardment

Since its development in the mid-1980s, microprojectile bombardment has been widely employed as a method for direct gene transfer into a wide range of plants, including the previously difficult-to-transform monocotyledonous species. Although the numerous instruments available for microprojectile-mediated gene delivery and their applications have been widely discussed, less attention has been paid to the critical factors which affect the efficiency of this method of gene delivery. In this review we do not wish to describe the array of devices used for microprojectile delivery or their uses which have already been definitively described, but instead wish to report on research developments investigating the factors which affect microprojectile-mediated transformation of plants.     

T—DNA介导的基因诱捕技术及其在植物功能基因组学研究中的应用

T-DNA介导的基因诱捕技术是近年来发展起来的鉴定和分离基因的方法。在拟南芥和水稻基因组测序已经完成的今天,该技术将在两者基因功能的研究中扮演举足轻重的角色。本文就T—DNA介导的基因诱捕系统、基因克隆和突变体库构建的研究进展及其在植物功能基因组学上的应用等内容进行了综述,并讨论了该技术应用中的一些问题。     

蓝藻基因转移系统的选择与建立

蓝藻是一类进行光合放氧的原核生物 ,因其结构的特殊性 ,已成为表达外源基因的理想宿主之一。然而外源基因转化系统的选择与建立一直影响着蓝藻基因工程的快速发展。总结了各类蓝藻基因转移系统的特点、影响因素、各系统间的优缺点、以及不同蓝藻株系最适基因转移系统的选择等 ,为利用蓝藻进行遗传操作提供可能 ,为蓝藻基因工程发展提供信息。     

Web Tools for Rice Transcriptome Analyses

Gene expression databases provide profiling data for the expression of thousands of genes to researchers worldwide. Oligonucleotide microarray technology is a useful tool that has been employed to produce gene expression profiles in most species. In rice, there are five genome-wide DNA microarray platforms: NSF 45K, BGI/Yale 60K, Affymetrix, Agilent Rice 44K, and NimbleGen 390K. Presently, more than 1,700 hybridizations of microarray gene expression data are available from public microarray depositing databases such as NCBI gene expression omnibus and Arrayexpress at EBI. More processing or reformatting of public gene expression data is required for further applications or analyses. Web-based databases for expression meta-analyses are useful for guiding researchers in designing relevant research schemes. In this review, we summarize various databases for expression meta-analyses of rice genes and web tools for further applications, such as the development of co-expression network or functional gene network.