Characterization of gene expression profiles in early bovine pregnancy using a custom cDNA microarray

Gene expression analysis comparing nonpregnant with pregnant bovine uteri, including placenta, was performed with a custom cDNA microarray containing 1,933 independent genes. These genes were classified into six categories according to biological function, as follows: cell and tissue structural dynamics (108 genes), intercellular communication (221), intracellular metabolism (265), cell cycle and apoptosis (26), regulation of gene expression (113), expressed sequence tag (EST) and function unknown (617), and uncomplemented genes (583 clones). This array possessed bovine placental/endometrial specificity, as it included many pregnancy-specific molecules, such as pregnancy-associated glycoprotein-1 (PAGs), placental lactogen (PLs), and prolactin-related protein-1 (PRPs). A total of 77 genes were induced and 12 repressed in the placenta/endometrium. Our results point to a fundamental role for bovine placental-specific genes such as PAGs, PLs, and PRPs, in implantation and placentogenesis, and document that cDNA microarray analysis from bovine placenta/endometrium is possible and is a specific tool for monitoring genome-wide gene expression during the establishment and maintenance of pregnancy.     

New experimental and computational approaches to the analysis of gene expression.

Public and private EST (Expressed Sequence Tag) programs provide access to a large number of ESTs from a number of plant species, including Arabidopsis, corn, soybean, rice, wheat. In addition to the homology of each EST to genes in GenBank, information about homology to all other ESTs in the data base can be obtained. To estimate expression levels of genes represented in the DuPont EST data base we count the number of times each gene has been seen in different cDNA libraries, from different tissues, developmental stages or induction conditions. This quantitation of message levels is quite accurate for highly expressed messages and, unlike conventional Northern blots, allows comparison of expression levels between different genes. Lists of most highly expresses genes in different libraries can be compiled. Also, if EST data is available for cDNA libraries derived from different developmental stages, gene expression profiles across development can be assembled. We present an example of such a profile for soybean seed development. Gene expression data obtained from Electronic Northern analysis can be confirmed and extended beyond the realm of highly expressed genes by using high density DNA arrays. The ESTs identified as interesting can be arrayed on nylon or glass and probed with total labeled cDNA first strand from the tissue of interest. Two-color fluorescent labeling allows accurate mRNA ratio measurements. We are currently using the DNA array technology to study chemical induction of gene expression and the biosynthesis of oil, carbohydrate and protein in developing seeds.     

Validation of rat reference genes for improved quantitative gene expression analysis using low density arrays

Real-time PCR has become increasingly important in gene expression profiling research, and it is widely agreed that normalized data are required for accurate estimates of messenger RNA (mRNA) expression. With increased gene expression profiling in preclinical research and toxicogenomics, a need for reference genes in the rat has emerged, and the studies in this area have not yet been thoroughly evaluated. The purpose of our study was to evaluate a panel of rat reference genes for variation of gene expression in different tissue types. We selected 48 known target genes based on their putative invariability. The gene expression of all targets was examined in 11 types of rat tissues using TaqMan low density array (LDA) technology. The variability of each gene was assessed using a two-step statistical model. The analysis of mean expression using multiple reference genes was shown to provide accurate and reliable normalized expression data. The least five variable genes from each specific tissue were recommended for future tissue-specific studies. Finally, a subset of investigated rat reference genes showing the least variation is recommended for further evaluation using the LDA platform. Our work should considerably enhance a researcher's ability to simply and efficiently identify appropriate reference genes for given experiments.     

Identification of differentially expressed genes in subcutaneous adipose tissue from subjects with familial combined hyperlipidemia

Subjects with familial combined hyperlipidemia (FCHL) are characterized by a complex metabolic phenotype with hyperlipidemia, insulin resistance, and central obesity. FCHL is due to impaired adipose tissue function superimposed on hepatic overproduction of lipoproteins. We investigated adipose tissue as an interesting target tissue for differential gene expression in FCHL. Human cDNA expression array analyses, in which adipose tissue from five FCHL patients was compared with that from four age, gender, and BMI matched controls, resulted in the identification of 22 up-regulated and three down-regulated genes. The genes differentially expressed imply activation of the adipocyte cell cycle genes. Furthermore, the differential expression of the genes coding for tumor necrosis factor alpha, interleukin 6, and intracellular adhesion molecule 1 support a role for adipose tissue in insulin resistance in FCHL subjects. The observed changes represent a primary genetic defect, an adaptive response, or a contribution of both.     

人胚鼻咽组织基因表达谱

以水囊引产５、６、７、８个月人胚胎鼻咽组织总ＲＮＡ逆转录标记ｃＤＮＡ探针,与代表５８８个基因的Ａｔｌａｓ＾ＴＭｃＤＮＡ阵列进行杂交,观察了这些基因在不同发育时期内的表达差异。结果发现与细胞分裂增殖及细胞生长相关的基因明显高表达,不同胎龄存在多个表达水平不同的基因及同一基因在不同时期表达水平也不一样,如早期生长反应蛋白１（ｅａｒｌｙ ｇｒｏｗｔｈ ｒｅｓｐｏｎｓｅ ｐｒｏｔｅｉｎ１）基因ＥＧＲＰ１在     

cDNA微阵列技术研究干旱胁迫下星星草基因的表达

运用cDNA微阵列技术研究干旱胁迫下星星草基因的表达。制备了载有660条星星草单一基因的cDNA微阵列。分别对干旱胁迫和对照星星草的mRNA进行荧光标记,并与载有星星草基因的cDNA微阵列进行杂交,通过芯片的杂交信号强度分析,共获得22个下调表达和17个上调表达的基因。BLASTX分析表明这些基因按功能可以分为脱水保护、信号转导与调控、活性氧清除、代谢、核糖体蛋白等几大类。发现了一些与干旱胁迫相关的功能未知基因和新基因。     

Partial Genome Scale Analysis of Gene Expression in Human Adipose Tissue Using DNA Array

Objective: Large scale analysis of gene expression in adipose tissue provides a basis for the identification of novel candidate genes involved in the pathophysiology of obesity. Our goal was to explore gene expression in human adipose tissue at a partial genome scale using DNA array. Research Methods and Procedures: Labeled cDNA, derived from human adipose tissue poly(A⁺) RNA, was hybridized to a DNA array containing over 18,000 human expressed sequence‐tagged (EST) clones. The results were analyzed by database searches. Results: Homology searches of the 300 EST clones with highest hybridization signals revealed that 145 contained DNA sequences identical to known genes and 79 could be linked to UniGene clusters. Of the 145 identified genes, 136 were nonredundant and subsequently characterized with respect to function and chromosomal localization by searching MEDLINE, UniGene, GeneMap, OMIM, SWISS‐PROT, the Genome Database, and the Location Data Base. The identified genes were grouped according to their putative functions; cell/organism defense (9.6%), cell division (5.1%), cell signaling/communication (19.8%), cell structure/motility (12.5%), gene/protein expression (16.9%), metabolism (16.2%), and unclassified (19.8%). Less than 50% of these genes have previously been reported to be expressed in adipose tissue. The chromosomal localization of 268 genes strongly expressed in adipose tissue showed that their relative abundance was significantly increased on chromosomes 11, 19, and 22 compared to the expected distribution of the same number of random genes. Discussion: Our study resulted in the identification of numerous genes previously not reported to be expressed in adipose tissue. These results suggest that DNA array is a powerful tool in the search for novel regulatory pathways within adipose tissue on a scale that is not possible using conventional methods.     

初探E2F1 对基因组内跨膜信号转导基因的调控作用

目的:从基因组全局性角度研究E2F1对包括离子通道和G蛋白偶联受体在内的重要的跨膜信号转导基因的调控作用。方法:对从TRED和IUPHAR获取的E2F1靶基因数据和基因组离子通道及G蛋白偶联受体基因数据进行数据联配,获取E2F1调控的离子通道基因(ICG)和G蛋白偶联受体基因,并对调控基因进行家族富集性分析和组织特异性分析。结果:发现E2F1对7个ICG具有调控作用,且具有钾离子通道富集性,调控的离子通道基因具有心脏、脑、消化系统组织表达特异性。获得的11个受E2F1调控的G蛋白偶联受体基因家族富集性不明显,组织特异性表达不一致。结论:E2F1可能通过对钾离子通道基因的表达调控,实现对相应组织的作用机理影响,相应调控作用的紊乱也将导致心脏、脑或消化系统疾病,但难以确定E2F1对GPCR的调控作用效果。     

Global gene expression profiles reveal an increase in mRNA levels of collagens, MMPs, and TIMPs in late radiation enteritis

Radiation enteritis, a common complication of radiation therapy for abdominal and pelvic cancers, is characterized by severe transmural fibrosis associated with mesenchymal cell activation, tissue disorganization, and deposition of fibrillar collagen. To investigate the mechanisms involved in this pathological accumulation of extracellular matrix, we studied gene expression of matrix components along with that of genes involved in matrix remodeling, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Hybrid selection on high-density cDNA array, real-time RT-PCR, gelatin zymography and immunohistochemistry were used to characterize the mRNA expression profile, activity, and tissue location of extracellular matrix-related genes in radiation enteritis compared with healthy ileum. cDNA array analysis revealed a strong induction of genes coding for collagens I, III, IV, VI, and VIII, SPARC, and tenascin-C, extracellular-matrix degrading enzymes (MMP-1, -2, -3, -14, -18+19), and metalloproteinase inhibitors (TIMP-1, -2, plasminogen activator inhibitor-1) in radiation enteritis. This increase was correlated with the degree of infiltration of the mucosa by inflammatory cells, and the presence of differentiated mesenchymal cells in the submucosa and muscularis propria. Despite the fact that expression of collagens, MMPs, and TIMPs simultaneously increase, quantification of net collagen deposition shows an overall accumulation of collagen. Our results indicate that late radiation enteritis tissues are subjected to active process of fibrogenesis as well as fibrolysis, with a balance toward fibrogenesis. This demonstrates that established fibrotic tissue is not scarred fixed tissue but is subjected to a dynamic remodeling process.     

Differential gene expression profile between PC-14 cells treated with free cisplatin and cisplatin-incorporated polymeric micelles

Cisplatin (CDDP)-incorporated polymeric micelles (CDDP/m) are a macromolecular carrier system possessing a time-modulated decaying property accompanied by sustained release of free drug. The gene expression profiles in nonsmall cell lung cancer PC-14 cells treated with free CDDP and CDDP/m were evaluated by a cDNA expression array for 807 genes. Although the total gene expression profile of the cells treated with CDDP/m approximated that of free CDDP in the hierarchical clustering analysis, a number of genes showed differential expression according to whether the cells had been treated with CDDP or CDDP/m. Ultimately, 50 genes with significant differential expression between cells treated with CDDP and CDDP/m were selected by principal component (PC) analysis and the unpaired t-test. The genes selected, including genes related to cell cycle regulation, apoptosis-related proteins, detoxification, and DNA repair enzymes, were considered to be related to CDDP-induced cytotoxicity. Interestingly, CDDP/m down-regulated the genes encoding integrins and matrix metalloproteinases (MMPs), which play an integral role in tumor invasion, metastasis, and angiogenesis, whereas free CDDP up-regulated them. The results suggest that use of the macromolecular carriers may yield additional therapeutic effects over free drug.