Tiling array-driven elucidation of transcriptional structures based on maximum-likelihood and Markov models

Tiling arrays of high-density oligonucleotide probes spanning the entire genome are powerful tools for the discovery of new genes. However, it is difficult to determine the structure of the spliced product of a structurally unknown gene from noisy array signals only. Here we introduce a statistical method that estimates the precise splicing points and the exon/intron structure of a structurally unknown gene by maximizing the odds or the ratio of posterior probabilities of the structure under the observation of array signal intensities and nucleic acid sequences. Our method more accurately predicted the gene structures than the simple threshold-based method, and more correctly estimated the expression values of structurally unknown genes than the window-based method. It was observed that the Markov model contributed to the precision of splice points, and that the statistical significance of expression (P-value) represented the reliability of the estimated gene structure and expression value well. We have implemented the method as a program ARTADE (ARabidopsis Tiling Array-based Detection of Exons) and applied it to the Arabidopsis thaliana whole-genome array data analysis. The database of the predicted results and the ARTADE program are available at http://omicspace.riken.jp/ARTADE/.     

Integrative analysis of genetical genomics data incorporating network structures

In a living organism, tens of thousands of genes are expressed and interact with each other to achieve necessary cellular functions. Gene regulatory networks contain information on regulatory mechanisms and the functions of gene expressions. Thus, incorporating network structures, discerned either through biological experiments or statistical estimations, could potentially increase the selection and estimation accuracy of genes associated with a phenotype of interest. Here, we considered a gene selection problem using gene expression data and the graphical structures found in gene networks. Because gene expression measurements are intermediate phenotypes between a trait and its associated genes, we adopted an instrumental variable regression approach. We treated genetic variants as instrumental variables to address the endogeneity issue. We proposed a two‐step estimation procedure. In the first step, we applied the LASSO algorithm to estimate the effects of genetic variants on gene expression measurements. In the second step, the projected expression measurements obtained from the first step were treated as input variables. A graph‐constrained regularization method was adopted to improve the efficiency of gene selection and estimation. We theoretically showed the selection consistency of the estimation method and derived the bound of the estimates. Simulation and real data analyses were conducted to demonstrate the effectiveness of our method and to compare it with its counterparts.     

Methodological optimization of polyethylenimine (PEI)-based gene delivery to the lungs of mice via aerosol application

BACKGROUND: Polyethylenimine (PEI) has been successfully used for gene delivery to the lungs of mice via aerosol application using a whole body nebulization device. In this report we optimized the design of such an aerosol device. METHODS: Aerosol devices were constructed as either serial inhalation apparatus or as a whole body nebulization chamber connected to an aerosol spacer placed in a horizontal or vertical position. PEI-based gene vectors were nebulized using a standard jet nebulizer and luciferase gene expression of various tissues was examined. RESULTS: Using a whole body aerosol device resulted in luciferase gene expression in the lungs of mice at the same level as compared with a serial inhalation apparatus. Whereas gene expression was enhanced in the presence of 5% CO(2)-in-air, anesthesia of mice strongly decreased gene expression even when mice were intubated with an intravascular cannula. Reduction of the median mass aerodynamic diameter (MMAD) of the aerosol from 3.4 to 0.27 microm by interposition of an aerosol spacer increased gene expression significantly 3-fold. Drying of the aerosol by silica gel additionally increased gene delivery significantly 3-fold. Reporter gene expression mediated by branched PEI 25 kDa was 9- and 15-fold higher as compared with linear PEIs of 22 and 25 kDa, respectively, and was dependent on the DNA concentration. Gene expression was detectable as soon as 6 h after gene vector application and reached a maximum after 72 h but was still detectable after 14 days. The presence of Zn(2+) did not increase gene expression. CONCLUSION: We propose aerosol drying as a novel and simple method of optimizing PEI-based gene delivery to the lungs.     

拟南芥AtcpSecA基因表达的特异性

前期研究表明AtcpSecA基因的突变使叶绿体发育缺陷,内部缺少正常类囊体片层结构,叶片呈黄白色。在此基础上我们进一步研究AtcpSecA基因的表达特异性,并构建了AtcpSecA基因启动子与报告基因GUS的融合基因AtcpSecA：：GUS,以农杆菌介导方法转化获得转基因拟南芥。GUS组织化学染色结果表明,在AtcpSecA：：GUS转基因拟南芥的下胚轴、子叶、叶片、果柄等绿色组织中有很强的GUS活性,而在根、花序和种荚等非绿色组织中几乎没有GUS活性。降低培养基中琼脂浓度转基因拟南芥中AtcpSecA：：GUS基因的表达明显受抑制,暗中则显著受到促进。     

2.45 GHz radiofrequency fields alter gene expression in cultured human cells

The biological effect of radiofrequency (RF) fields remains controversial. We address this issue by examining whether RF fields can cause changes in gene expression. We used the pulsed RF fields at a frequency of 2.45 GHz that is commonly used in telecommunication to expose cultured human HL-60 cells. We used the serial analysis of gene expression (SAGE) method to measure the RF effect on gene expression at the genome level. We observed that 221 genes altered their expression after a 2-h exposure. The number of affected genes increased to 759 after a 6-h exposure. Functional classification of the affected genes reveals that apoptosis-related genes were among the upregulated ones and the cell cycle genes among the downregulated ones. We observed no significant increase in the expression of heat shock genes. These results indicate that the RF fields at 2.45 GHz can alter gene expression in cultured human cells through non-thermal mechanism.     

普通烟草ARF基因家族序列的鉴定与表达分析

ARF(AUXIN RESPONSE FACTOR)基因含有一个B3功能域和具有转录激活或抑制活性的中心功能域,在植物发育过程中起到非常重要的作用。本研究采用生物信息学方法,根据拟南芥ARF基因序列鉴定了普通烟草基因组中的ARF基因,并对家族成员进行了序列特征、系统发生、亚细胞定位和表达模式分析。目前在普通烟草基因组中共得到50个ARF基因成员,其基因结构相对复杂,一般含有10个外显子。亚细胞定位结果表明,少数ARF蛋白定位到线粒体或叶绿体,大多数未检测到定位信号。转录组数据分析表明,ARF基因具有不同的组织表达模式,部分基因表现出组织特异性。这些研究结果为普通烟草ARF基因家族功能的深入研究奠定了基础。     

基于基因表达谱的疾病亚型特征基因挖掘方法

在本研究中,提出了一种基于基因表达谱的疾病亚型特征基因挖掘方法,该方法基于过滤后基因表达谱,融合无监督聚类识别疾病亚型技术和提出的衡量特征基因对疾病亚型鉴别能力的模式质量测度,以嵌入的方式实现特征基因挖掘。最后将提出的方法应用于40例结肠癌组织与22例正常结肠组织中2000个基因的表达谱实验数据,结果显示:提出的方法是一种可行的疾病亚型特征基因挖掘方法,方法的优势在于可并行实现疾病亚型划分和特征基因识别。     

A fast and efficient method for transiently transfecting ES cells: application to the development of systems for conditional gene expression

Classically, mouse embryonic stem (ES) cells are transfected by electroporation, a method that requires a large number of cells. Here we describe a protocol using a liposome based transfection agent that is a very simple, rapid and cost effective way of transiently transfecting very low numbers of ES cells. We found this method very useful in screening a large number of ES clones when working with inducible expression systems in which at least two elements are required for regulated expression of the gene of interest. After stable transfection of the first component, clones can be easily and rapidly screened for expression of the gene of interest by transiently transfecting the second component of the system using this protocol.     

Rank of Correlation Coefficient as a Comparable Measure for Biological Significance of Gene Coexpression

Information regarding gene coexpression is useful to predict gene function. Several databases have been constructed for gene coexpression in model organisms based on a large amount of publicly available gene expression data measured by GeneChip platforms. In these databases, Pearson''s correlation coefficients (PCCs) of gene expression patterns are widely used as a measure of gene coexpression. Although the coexpression measure or GeneChip summarization method affects the performance of the gene coexpression database, previous studies for these calculation procedures were tested with only a small number of samples and a particular species. To evaluate the effectiveness of coexpression measures, assessments with large-scale microarray data are required. We first examined characteristics of PCC and found that the optimal PCC threshold to retrieve functionally related genes was affected by the method of gene expression database construction and the target gene function. In addition, we found that this problem could be overcome when we used correlation ranks instead of correlation values. This observation was evaluated by large-scale gene expression data for four species: Arabidopsis, human, mouse and rat.     

Gene Expression in Tilapia Following Oral Delivery of Chitosan-Encapsulated Plasmid DNA Incorporated into Fish Feeds

DNA delivery into fish is important for transient gene expression, (e.g., DNA vaccination). Previous studies have generally focused on intramuscular injection of DNA vaccines into fish. However, this method is obviously impractical and laborious for injecting large numbers of fishes. This study reports oral delivery of a construct expressing the β-galactosidase reporter gene into fish by encapsulating the DNA in chitosan and incorporating it into fish feeds. We found that β-galactosidase expression could be observed in the stomachs, spleens, and gills of fishes fed with flakes containing the chitosan-DNA complex. These results suggest that DNA vaccines and other constructs can be easily and cheaply delivered into fishes orally by use of carriers and incorporation into fish feeds.     

Delivery of electric pulses for DNA electrotransfer to mouse muscle does not induce the expression of stress related genes

In vivo gene transfer to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic delivery of therapeutic proteins. Electrotransfer is a powerful method for DNA transfer into skeletal muscle. In view of the broad potential gene therapy clinical application of electrotransfer offers, it is important to perform toxicology studies on electrotransfered muscle tissue. We have investigated if the delivery of square wave electric pulses of low field strength and long duration to mouse tibial cranial muscle induced the expression of stress related genes. We have profiled gene expression patterns in muscles at different times after delivery of electric pulses using Stress/Toxicology microarrays. No significant variation in the expression of stress related-genes was detected between treated and non-treated muscles. This suggests that application of adequate, fine-tuned, electric pulses to the skeletal muscle is a non-toxic technique for gene therapy.     

Gene profiling of hippocampal neuronal culture

We performed mRNA expression profiling of mouse primary hippocampal neurones undergoing differentiation in vitro. We show that 2314 genes significantly changed expression during neuronal differentiation. The temporal resolution of our experiment (six time points) permits us to distinguish between gene expression patterns characteristic for the axonal and for the dendritic stages of neurite outgrowth. Cluster analysis reveals that, in the process of in vitro neuronal differentiation, a high level of expression of genes involved in the synthesis of DNA and proteins precedes the up regulation of genes involved in protein transport, energy generation and synaptic functions. We report in detail changes in gene expression for genes involved in the synaptic vesicle cycle. Data for other genes can be accessed at our website. We directly compare expression of 475 genes in the differentiating neurones and the developing mouse hippocampus. We demonstrate that the program of gene expression is accelerated in vitro as compared to the situation in vivo. When this factor is accounted for, the gene expression profiles in vitro and in vivo become very similar (median gene-wise correlation 0.787). Apparently once the cells have taken a neuronal fate, the further program of gene expression is largely independent of histological or anatomical context. Our results also demonstrate that a comparison across the two experimental platforms (cDNA microarrays and oligonucleotide chips) and across different biological paradigms is feasible.     

Genomic studies of gene expression: regulation of the Wilson disease gene

Bacterial artificial chromosomes (BACs) have many advantages over other large-insert cloning vectors and have been used for a variety of genetic applications, including the final contigs of the human genome. We describe the utilization of a BAC construct to study gene regulation in a tissue culture-based system, using a 170-kb clone containing the entire Wilson disease (WND) locus as a model. A second BAC construct that lacked a putative negatively regulating promoter sequence was created. A nonviral method of gene delivery was applied to transfect three human cell lines stably with each construct. Our results show correct WND gene expression from the recombinant locus and quantification revealed significantly increased expression from the clone lacking the negative regulator. Comparison with conventional methods confirms the reliability of the genomic approach for thorough examination of gene expression. This experimental system illustrates the potential of BAC clones in genomic gene expression studies, new gene therapy strategies, and validation of potential molecular targets for drug discovery.     

应用基因芯片筛选鼻咽癌信号转导相关基因

目的：建立具有组织特异性的鼻咽癌基因表达谱,筛选鼻咽癌中信号转导相关基因。方法：采用深圳微芯公司基于玻片的包含8046个人类基因的基因芯片,检测7例鼻咽癌组织及1例鼻咽炎组织,初步获得鼻咽癌异常表达基因;结合GO分类从异常表达的基因中筛选信号转导相关基因,以Biocarta信号通路数据库查询筛选基因相关转导信号通路信息。结果：在鼻咽癌组织独得1241个异常用表达基因,其中高表达基因871个,低表达基因343个。发现28个差异表达基因与细胞的信号转导相关,其中表达上调的21个,表达下调的7个。结论：成功建立了具有组织特异性的鼻咽癌基因表达谱,初步获得了鼻咽癌信号转导相关基因。     

PlantRGS: a web server for the identification of most suitable candidate reference genes for quantitative gene expression studies in plants

Normalization of quantitative gene expression data with a suitable reference gene is essential for accurate and reliable results. However, the availability and choice of most suitable reference gene(s) showing uniform expression across all the experimental conditions remain a drawback. We have developed a web server, PlantRGS (http://www.nipgr.res.in/PlantRGS), for the identification of most suitable candidate reference gene(s) at the whole-genome level using microarray data for quantitative gene expression studies in plants. Microarray data from more than 11 000 tissue samples for nine plant species have been included in the PlantRGS for meta-analysis. The web server provides a user-friendly graphical user interface-based analysis tool for the identification of most suitable reference genes in the selected plant species under user-defined experimental conditions. Various parameter options and output formats will help users to investigate desired number of most suitable reference genes with wide range of expression levels. Validation of results revealed that novel reference genes identified by the PlantRGS outperforms the traditionally used reference genes in terms of expression stability. We anticipate that the PlantRGS will provide a platform for the identification of most suitable reference gene(s) under given experimental conditions and facilitate quantitative gene expression studies in plants.