Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein 9(CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs(sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies.Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.     

Virus-induced gene silencing: A versatile tool for discovery of gene functions in plants

Virus-induced gene silencing (VIGS) is a technology that exploits an antiviral defense mechanism in plants as a tool for plant reverse genetics. VIGS circumvents the need for plant transformation, is methodologically simple and yields rapid results. Various VIGS vectors have been developed and have helped to unravel the functions of genes involved in processes such as disease resistance, abiotic stress, cellular signaling and secondary metabolite biosynthesis.     

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．     

腺病毒载体在生物治疗中的应用

腺病毒载体是目前重要的基因转移载体之一。腺病毒可作为真核基因表达载体,可制成灭活、重组或抗癌疫苗用于预防呼吸道疾病、癌症和肝炎等传染病。在癌症的基因治疗方面,Ａｄ 载体可运载肿瘤抑制基因,自身基因编码蛋白能诱导细胞调亡,可作为前药物感染细胞,还能利用Ａｄ 的一些特殊复制子,达到治疗肿瘤的目的     

A cardiovascular EST repertoire: progress and promise for understanding cardiovascular disease

The application of expressed sequence tag (EST) technology has proven to be an effective tool for gene discovery and the generation of gene expression profiles. The generation of an EST resource for the cardiovascular system has revealed significant insights into the changes in gene expression that guide heart development and disease. Furthermore, an important genetic resource has been developed for cardiovascular biology that is valuable for data mining and disease gene discovery.     

New developments in the analysis of gene expression

An understanding of the relationship between gene expression, protein expression and the influences of genetic responses upon gene function is vital before we can understand the complexity of genomes. Traditional methods for the study of gene expression are limited to studying small groups of genes at a time and a source of pure starting material has been difficult to obtain. Recent technological advances have enabled large numbers of genes, from specific cell populations, to be studied in a single experiment. Laser capture microdissection (LCM) and microarray technology are providing the next revolution in the study of gene expression. LCM-based molecular analysis of histopathological lesions can be applied to any disease process that is accessible through tissue sampling. Examples include: (i) mapping the field of genetic changes associated with oxidative stress; (ii) analysis of gene expression patterns in atherosclerotic tissues, sites of inflammation and Alzheimer's disease plaques; (iii) infectious micro-organism diagnosis; and (iv) typing of cells within disease foci. Microarray hybridisation glass chips spotted with sets of genes can then be used to obtain a molecular fingerprint of gene expression in the microdissected cells. The variation of expressed genes or alterations in the cellular DNA that correlate with a particular disease state can be compared within or between individual samples. The identification of gene expression patterns may provide vital information for the understanding of the disease process and may contribute to diagnostic decisions and therapies tailored to the individual patient. Molecules found to be associated with defined pathological lesions may provide clues about new therapeutic targets in the future.     

New developments in the analysis of gene expression

Abstract

An understanding of the relationship between gene expression, protein expression and the influences of genetic responses upon gene function is vital before we can understand the complexity of genomes. Traditional methods for the study of gene expression are limited to studying small groups of genes at a time and a source of pure starting material has been difficult to obtain. Recent technological advances have enabled large numbers of genes, from specific cell populations, to be studied in a single experiment. Laser capture microdissection (LCM) and microarray technology are providing the next revolution in the study of gene expression. LCM-based molecular analysis of histpathological lesions can be applied to any disease process that is accessible through tissue sampling. Examples include: (i) mapping the field of genetic changes associated with oxidative stress; (ii) analysis of gene expression patterns in atherosclerotic tissues, sites of inflammation and Alzheimer's disease plaques; (iii) infectious micro-organism diagnosis; and (iv) typing of cells within disease foci. Microarray hybridisation glass chips spotted with sets of genes can then be used to obtain a molecular fingerprint of gene expression in the microdissected cells. The variation of expressed genes or alterations in the cellular DNA that correlate with a particular disease state can be compared within or between individual samples. The identification of gene expression patterns may provide vital information for the understanding of the disease process and may contribute to diagnostic decisions and therapies tailored to the individual patient. Molecules found to be associated with defined pathological lesions may provide clues about new therapeutic targets in the future.     

Cystic fibrosis transmembrane conductance regulator and the etiology and pathogenesis of cystic fibrosis.

Cystic fibrosis (CF) is an inherited disorder causing pancreatic, pulmonary, and sinus disease in children and young adults. Abnormal viscosity of mucous secretions is a hallmark of the disease, and is believed to be the result of altered electrolyte transport across epithelial cell membranes. The monogenic etiology of this disease has been apparent for more than 40 years, but the defective gene has only recently been identified. This was made possible because of a revolution in genetic technology, called positional cloning, which can pinpoint disease genes without previous knowledge of the abnormal protein product. The protein encoded by the gene defective in CF has been termed the CF transmembrane conductance regulator (CFTR) because of its postulated role in electrolyte transport. Studies investigating the normal function of CFTR and how mutations affect that function, thereby causing CF, have required the combined skills of clinicians, geneticists, molecular biologists, and physiologists. From this collaborative effort a greater understanding of the pathogenesis of this disorder is now emerging. It may soon be possible to introduce novel therapies derived from this new knowledge that will be aimed directly at the basic defect. An ever-increasing number of genes of unknown function will be identified by continuing advances in molecular genetic technology and the advent of the genome sequencing project. The experience in cystic fibrosis research may prove to be a paradigm for investigation of the function of genes isolated by positional cloning methods.     

Virus-induced gene silencing in plants

Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying inserts derived from host genes the process can be additionally targeted against the corresponding mRNAs. VIGS has been used widely in plants for analysis of gene function and has been adapted for high-throughput functional genomics. Until now most applications of VIGS have been in Nicotiana benthamiana. However, new vector systems and methods are being developed that could be used in other plants, including Arabidopsis. Here we discuss practical and theoretical issues that are specific to VIGS rather than other gene "knock down" or "knockout" approaches to gene function. We also describe currently used protocols that have allowed us to apply VIGS to the identification of genes required for disease resistance in plants. These methods and the underlying general principles also apply when VIGS is used in the analysis of other aspects of plant biology.     

Transgenic animals in inflammatory disease models

Inflammatory diseases affect a significant portion of the population worldwide and have been intensely studied for several decades. The advent of transgenic technology has allowed researchers to study individual gene contributions to the pathogenesis of these diseases. This has been done using standard inflammatory disease models in transgenic animals and by identifying novel models through the spontaneous generation of disease in the transgenic animal. Recent advances have been made in the understanding of rheumatoid arthritis, pulmonary inflammation, multiple sclerosis and inflammatory bowel disease through the use of transgenic animals in models of human inflammatory disease.     

转人工miRNA抗玉米粗缩病毒载体玉米的培育及其抗病能力

玉米是重要的粮食作物,水稻黑条矮缩病毒(RBSDV)是玉米粗缩病的病原,由其引起的玉米粗缩病给玉米生产造成重大损失。利用人工mi RNA构建抗病毒植物的技术已经在多种植物中被证明有效,但是在玉米中的尝试未见报道。实验根据玉米zea-mi R159a的前体序列和RBSDV基因组中编码功能蛋白的基因和基因沉默抑制子的序列信息设计引物,构建了用于沉默RBSDV编码基因和基因沉默抑制子的ami RNA(Artificial mi RNA)基因。构建p CAMBIA3301-121-ami RNA植物表达载体,利用农杆菌介导法转化玉米自交系综31(Z31)。对转基因玉米进行分子检测,选择mi RNA表达量高的纯合体株系进行自然发病实验,按0-4的分级标准调查玉米粗缩病的严重度。结果表明,转抗粗缩病毒人工mi RNA载体玉米纯合体株系的抗病表现好于野生型玉米,其中针对基因组6的S6-mi R159转基因玉米抗病情况较好。研究表明利用人工mi RNA技术构建抗病毒病玉米新品种是可行的。     

基因芯片技术检测细菌耐药性的研究进展

基因芯片技术是将无数预先设计好的寡核苷酸、cDNA、基因组 (Genomic)DNA在芯片上做成点阵 ,与样品中同源核酸分子杂交 ,对样品的序列信息进行高效的解读和分析 ,大规模获取相关生物信息。该技术应用领域主要有表达谱分析、基因突变及多态性分析、疾病诊断和预测、DNA测序、药物筛选、检测筛选耐药基因、微生物菌种鉴定及致病机制研究等。着重介绍了基因芯片技术检测细菌耐药性方面的国外研究进展。基因芯片可以大量、快捷地检测出细菌耐药性菌株以及引起细菌耐药性的基因的突变 ,由于其在检测中的高效率 ,因此要优越于传统的细菌学检测技术。基因芯片技术在细菌耐药性检测中有着巨大的应用价值 ,具有广阔的应用前景。     

Designing gene delivery vectors for cardiovascular gene therapy

Genetic therapy in the cardiovascular system has been proposed for a variety of diseases ranging from prevention of vein graft failure to hypertension. Such diversity in pathogenesis requires the delivery of therapeutic genes to diverse cell types in vivo for varying lengths of time if efficient clinical therapies are to be developed. Data from extensive preclinical studies have been compiled and a certain areas have seen translation into large-scale clinical trials, with some encouraging reports. It is clear that progress within a number of disease areas is limited by a lack of suitable gene delivery vector systems through which to deliver therapeutic genes to the target site in an efficient, non-toxic manner. In general, currently available systems, including non-viral systems and viral vectors such as adenovirus (Ad) or adeno-associated virus (AAV), have a propensity to transduce non-vascular tissue with greater ease than vascular cells thereby limiting their application in cardiovascular disease. This problem has led to the development and testing of improved vector systems for cardiovascular gene delivery. Traditional viral and non-viral systems are being engineered to increase their efficiency of vascular cell transduction and diminish their affinity for other cell types through manipulation of vector:cell binding and the use of cell-selective promoters. It is envisaged that future use of such technology will substantially increase the efficacy of cardiovascular gene therapy.     

基于RNA的抗HIV-1基因治疗方法研究进展

艾滋病自发现以来在全球范围内迅速蔓延,危害性极高,目前广泛采用的高效抗逆转录病毒疗法(HAART)虽能够显著提高HIV-1感染者生活质量,但存在着价格昂贵,耐药和副作用的问题经常会导致HAART治疗的中断。要获得长期持续的抗病毒治疗效果还有待于研发新的抗病毒药物和治疗方法。近年来随着分子生物技术、干细胞研究、纳米技术等相关技术的发展,关于抗HIV-1基因治疗方法的研究受到了广泛关注。主要针对基于RNA的抗HIV-1基因治疗方法,包括反义RNA、核酶、RNA诱饵以及RNA干扰技术在抗HIV-1基因治疗方面进行综述。研究表明,以RNA为基础的抗HIV-1基因治疗方法有望成为传统治疗方法的一种有效辅助手段。     

Gene transfer into eukaryotic cells using activated polyamidoamine dendrimers

The development of efficient methods to transfer genes into eukaryotic cells is important for molecular biotechnology. A number of different technologies to mediate gene transfer have been developed over the last 35 years, but most have drawbacks such as cytotoxicity, low efficiency and/or restricted applicability. Activated polyamidoamine (PAMAM)-dendrimers provide a new technology for gene transfer that offers significant advantages over classical methods. Reagents based on this technology provide high gene transfer efficiencies, minimal cytotoxicity, and can be used with a broad range of cell types. This technology could also be useful for in vivo gene transfer in gene therapy applications.     

RNAi技术在昆虫学中的研究进展及展望

在昆虫中,RNAi是一种对抗外源病毒的天然免疫方式,基于生物体中的这种内在机制而建立的RNAi技术已经被广泛用来研究多种昆虫基因的功能。近年的研究结果表明RNAi技术在抵御害虫和防治益虫疾病方面具有潜在的应用价值,有可能对农业有害生物的控制起到巨大的推动作用。本文综述了RNAi与昆虫免疫、及其在昆虫基因功能研究、害虫控制、益虫疾病控制和昆虫系统生物学方面的最新研究进展,并展望了RNAi在昆虫学研究中的发展趋势。     

Microarrays in infection and immunity

Over the past decade, microarrays have revolutionized the scientific world as dramatically as the internet has changed everyday life. From the initial applications of DNA microarrays to uncover gene expression patterns that are diagnostic and prognostic of cancer, understanding the interplay between immune responses and disease has been a prime application of this technology. More recent efforts have moved beyond genetic analysis to functional analysis of the molecules involved, including identification of immunodominant antigens and peptides as well as the role of post-translational glycosylation. Here, we focus on recent applications of microarray technology in understanding the detailed chemical biology of immune responses to disease in an effort to guide development of vaccines and other protective therapies.     

Beyond knockout rats

The ability to “knockout” specific genes in mice via embryonic stem (ES) cell-based gene-targeting technology has significantly enriched our understanding of gene function in normal and disease phenotypes. Improvements on this original strategy have been developed to enable the manipulation of genomes in a more sophisticated fashion with unprecedented precision. The rat is the model of choice in many areas of scientific investigation despite the lack of rat genetic toolboxes. Most Recent advances of zinc finger nucleases (ZFNs) and rat ES cells are diminishing the gap between rat and mouse with respect to reverse genetic approaches. Importantly, the establishment of rat ES cell-based gene targeting technology, in combination with the unique advantages of using rats, provides new, exciting opportunities to create animal models that mimic human diseases more faithfully. We hereby report our recent results concerning finer genetic modifications in the rat, and propose their potential applications in addressing biological questions.     

DNA microarray analysis of the correlation between gene expression patterns and acquired resistance to 5-FU/cisplatin in gastric cancer

The mechanisms of intrinsic and/or acquired anti-cancer drug resistance have been described in in vitro resistance models, but the clinical relevance has remained undefined. We undertook a prospective study to identify correlations between gene expression and clinical resistance to 5-FU/cisplatin. We compared expression profiles from gastric cancer endoscopic biopsy specimens obtained at a chemosensitive state (partial remission after 5-FU/cisplatin) with those obtained at a refractory state (disease progression), using Affymetrix oligonucleotide microarray technology (U133A). Using 119 discriminating probes and a cross-validation approach, we were able to correctly identify the chemo-responsiveness of 7 pairs of training samples and 1 independent test pair. These exploratory data demonstrate that the gene expression profiles differ between chemosensitive and refractory state gastric cancer biopsy samples.