Immunosuppressive drug-free operational immune tolerance in human kidney transplants recipients. Part II. Non-statistical gene microarray analysis

Kidney transplant is the reference treatment for patients with end-stage renal disease, but patients may develop long-term rejection of the graft. However, some patients do not reject the transplant, but instead are operationally tolerant state despite withdrawal of immunosuppressive treatment. In this second article we outline a microarray-based identification of key leader genes associated respectively to rejection and to operational tolerance of the kidney transplant in humans by utilizing a non/statistical bioinformatic approach based on the identification of "key genes," either as those mostly changing their expression, or having the strongest interconnections. A uniquely informative picture emerges on the genes controlling the human transplant from the detailed comparison of these findings with the traditional statistical SAM (Tusher et al. 2001 Proc Natl Acad Sci USA 98:5116-5121) analysis of the microarrays and with the clinical study carried out in the accompanying part I article.     

AKT1 leader gene and downstream targets are involved in a rat model of kidney allograft tolerance

Tolerance is the so‐called “Holy Grail” of transplantation but achieving this state is proving a major challenge, particularly in the clinical settings. This tolerance state can be induced in rodent models using a variety of maneuvers. This phenomenon is classically characterized by donor specificity (recipients accept a secondary donor‐specific allograft but reject third‐party allograft) as well as by the absence of chronic rejection lesion. We previously showed that administration and anti‐donor anti‐class II serum on the day of transplantation induce tolerance to a kidney allograft in the LEW‐1W to LEW‐1A strain combination. In this study, we used DNA microarrays to compare gene patterns involved in anti‐donor anti‐class II tolerated or untreated syngeneic kidney transplants in this strain combination. Statistical and non‐statistical analyses were combined with ab initio analysis, using the recently developed leader gene approach, to shed new light on this phenomenon. Theoretical and experimental results suggest that tolerance and rejection outcome may be in large part determined by low expression variations of some genes, which can form a core gene network around specific genes such as Rac1, NFKB1, RelA, AKT1, IKBKB, BCL2, BCLX, and CHUK. Through this model, we showed that AKT1 gene, WNT pathway and NO synthesis are strictly connected to each other and may play an important role in kidney tolerance and rejection processes, with AKT1 gene being the center of this complex network of interactions. J. Cell. Biochem. 111: 709–719, 2010. © 2010 Wiley‐Liss, Inc.     

机械点样DNA微点阵技术及其在基因表达分析上的应用(I)

从芯片制作、芯片杂交、芯片扫读与图像分析、基因表达数据分析等方面,详细介绍了机械点样DNA微点阵技术及其应用于多基因表达分析的基本步骤与原理。     

Tissue-specific gene expression in soybean (Glycine max) detected by cDNA microarray analysis

We have constructed cDNA microarrays for soybean (Glycine maxL. Merrill), containing approximately 4,100 Unigene ESTs derived from axenic roots, to evaluate their application and utility for functional genomics of organ differentiation in legumes. We assessed microarray technology by conducting studies to evaluate the accuracy of microarray data and have found them to be both reliable and reproducible in repeat hybridisations. Several ESTs showed high levels (50 fold) of differential expression in either root or shoot tissue of soybean. A small number of physiologically interesting, and differentially expressed sequences found by microarray analysis were verified by both quantitative real-time RT-PCR and Northern blot analysis. There was a linear correlation (r² = 0.99, over 5 orders of magnitude) between microarray and quantitative real-time RT-PCR data. Microarray analysis of soybean has enormous potential not only for the discovery of new genes involved in tissue differentiation and function, but also to study the expression of previously characterised genes, gene networks and gene interactions in wild-type, mutant or transgenic plants.     

Catecholamines and angiotensinogen gene expression in kidney proximal tubular cells

To investigate the molecular mechanism(s) of action of catecholamines on the expression of the angiotensinogen (ANG) gene in kidney proximal tubular cells, we used opossum kidney (OK) cells with a fusion gene containing the 5-flanking regulatory sequence of the rat ANG gene fused with a human growth hormone (hGH) gene as a reporter, pOGH (rANG N-1498/+18), permanently integrated into their genomes. The level of expression of the ANG-GH fusion gene was quantified by the amount of immunoreactive-hGH (IR-hGH) secreted into the medium. The addition of norepinephrine (NE), isoproterenol (a ₁/₂-adrenergic receptor (AR) agonist) and iodoclonidine (an ₂-AR agonist) stimulated the expression of the ANG-GH fusion gene in a dose-dependent manner, whereas the addition of epinephrine and phenylephrine (₁-AR agonist) had no effect. The stimulatory effect of NE was blocked by the presence of propranolol (-AR blocker), atenolol (₁-AR blocker), yohimbine (₂-AR blocker), Rp-cAMP (an inhibitor of cAMP-dependent protein kinase AI & AII) and staurosporine (an inhibitor of protein kinase C), but was not blocked by ICI 118, 551 (₂-AR blocker) and prazosin (₁-AR blocker). The addition of a combination of isoproterenol and iodoclonidine or a combination of 8-Bromo-cAMP (8-Br-cAMP) and phorbol 12-myristate (PMA) synergistically stimulated the expression of the ANG-GH fusion gene as compared to the addition of isoproterenol, iodoclonidine, 8-Br-cAMP or PMA alone. Furthermore, the addition of NE, 8-Br-cAMP or PMA stimulated the expression of pOGH (rANG N-806/-779/-53/+18), a fusion gene containing the putative cAMP responsive element (CRE, ANG N-806/-779) upstream of the ANG promoter (ANG N-53/+18) in OK cells, but had no effect on the expression of fusion genes containing the mutant of the CRE. Gel mobility shift assays revealed that the ANG-CRE binds with the DNA-binding domain (bZIP 254-327) of the cAMP-responsive binding protein (CREB). The binding of the labeled ANG-CRE to CREB (bZIP254-327) was displaced by unlabeled ANG-CRE and the CRE of the somatostatin gene but not by the mutants of the ANG-CRE. Finally, NE stimulated the phosphorylation of CREB in OK cells. These studies demonstrate that the molecular mechanism(s) of NE action on the expression of the ANG gene in OK cells may be mediated via both the PKA and PKC signalling pathways and via the phosphorylation of CREB. The phosphorylated CREB then interacts with the CRE in the 5-flanking region of the ANG gene and subsequently stimulates the gene expression.     

Laser capture microdissection for the analysis of gene expression during embryogenesis of Arabidopsis

It is during embryogenesis that the body plan of the developing plant is established. Analysis of gene expression during embryogenesis has been limited due to the technical difficulty of accessing the developing embryo. Here we demonstrate that laser capture microdissection can be applied to the analysis of embryogenesis. We show how this technique can be used in concert with DNA microarray for the large-scale analysis of gene expression in apical and basal domains of the globular-stage and heart-stage embryo, respectively, when critical events of polarity, symmetry and biochemical differentiation are established. This high resolution spatial analysis shows that up to approximately 65% of the genome is expressed in the developing embryo, and that differential expression of a number of gene classes can be detected. We discuss the validity of this approach for the functional analysis of both published and previously uncharacterized essential genes.     

Microarray analysis of gene expression with age in individual nematodes

We compare the aging of wild-type and long-lived C. elegans by gene expression profiling of individual nematodes. Using a custom cDNA array, we have characterized the gene expression of 4-5 individuals at 4 distinct ages throughout the adult lifespan of wild-type N2 nematodes, and at the same ages for individuals of the long-lived strain daf-2(e1370). Using statistical tools developed for microarray data analysis, we identify genes that differentiate aging N2 from aging daf-2, as well as classes of genes that change with age in a similar way in both genotypes. Our novel approach of studying individual nematodes provides practical advantages, since it obviates the use of mutants or drugs to block reproduction, as well as the use of stressful mass-culturing procedures, that have been required for previous microarray studies of C. elegans. In addition, this approach has the potential to uncover the molecular variability between individuals of a population, variation that is missed when studying pools of thousands of individuals.     

Microarray-based expression analysis of human osteoblast-like cell response to anodized titanium surface

An anodized surface significantly enhanced the adhesion of human osteoblast-like MG-63 cells to titanium. Using cDNA microarray analysis, five genes were differentially expressed while the rest remained unaltered. The results demonstrated that the anodized surface enhances cellular adhesion without significantly affecting the pattern of gene expression.     

Microarray analysis of differential gene expression in sensitive and resistant pig to Escherichia coli F18

In this study, Agilent two‐colour microarray‐based gene expression profiling was used to detect differential gene expression in duodenal tissues collected from eight full‐sib pairs of Sutai pigs differing in adhesion phenotype (sensitivity and resistance to Escherichia coli F18). Using a two‐fold change minimum threshold, we found 18 genes that were differentially expressed (10 up‐regulated and eight down‐regulated) between the sensitive and resistant animal groups. Our gene ontology analysis revealed that these differentially expressed genes are involved in a variety of biological processes, including immune responses, extracellular modification (e.g. glycosylation), cell adhesion and signal transduction, all of which are related to the anabolic metabolism of glycolipids, as well as to inflammation‐ and immune‐related pathways. Based on the genes identified in the screen and the pathway analysis results, real‐time PCR was used to test the involvement of ST3GAL1 and A genes (of glycolipid‐related pathways), SLA‐1 and SLA‐3 genes (of inflammation‐ and immune‐related pathways), as well as the differential genes FUT1, TAP1 and SLA‐DQA. Subsequently, real‐time PCR was performed to validate seven differentially expressed genes screened out by the microarray approach, and sufficient consistency was observed between the two methods. The results support the conclusion that these genes are related to the E. coli F18 receptor and susceptibility to E. coli F18.     

Microarray analysis of gene expression profiles in the bovine mammary gland during lactation

Mammary glands undergo functional and metabolic changes during virgin, lactation and dry periods. A total of 122 genes were identified as differentially expressed, including 79 up-regulated and 43 down-regulated genes during lactation compared with virgin and dry periods. Gene ontology analysis showed the functional classification of the up-regulated genes in lactation, including transport, biosynthetic process, signal transduction, catalytic activity, immune system process, cell death, and positive regulation of the developmental process. Microarray data clarified molecular events in bovine mammary gland lactation.     

The microarray analysis for gene expression in haploids and diploids derived from twin-seedling rice

In this study, microarray technique was employed to analyze the gene expression at the RNA level between haploids and corresponding diploids derived from a rice twin-seedling line SARII-628. Differ- ent degrees of expression variations were observed in the plant after haploidization. The main results are as follows: (1) after haploidization, the ratio of the sensitive loci was 2.47% of the total loci designed on chip. Those loci were randomly distributed on the 12 pairs of rice chromosomes and the activated loci were more than the silenced ones. (2) Gene clusters on chromosome were observed for 33 se- quences. (3) GoPipe function classification for 575 sensitive loci revealed an involvement in the bio- logical process, cell component and molecular function. (4) RT-PCR generally validated the result from microarray with a coincidence rate of 83.78%. And for the randomly-selected activated or silenced loci in chip analysis, the coincidence rate was up to 91.86%.