Analysis of transcriptional profiles and functional clustering of global cerebellar gene expression in PCD3J mice

The Purkinje cell degeneration (PCD) mutant mouse is characterized by a degeneration of cerebellar Purkinje cells and progressive ataxia. To identify the molecular mechanisms that lead to the death of Purkinje neurons in PCD mice, we used Affymetrix microarray technology to compare cerebellar gene expression profiles in pcd3J mutant mice 14 days of age (prior to Purkinje cell loss) to unaffected littermates. Microarray analysis, Ingenuity Pathway Analysis (IPA) and expression analysis systematic explorer (EASE) software were used to identify biological and molecular pathways implicated in the progression of Purkinje cell degeneration. IPA analysis indicated that mutant pcd3J mice showed dysregulation of specific processes that may lead to Purkinje cell death, including several molecules known to control neuronal apoptosis such as Bad, CDK5 and PTEN. These findings demonstrate the usefulness of these powerful microarray analysis tools and have important implications for understanding the mechanisms of selective neuronal death and for developing therapeutic strategies to treat neurodegenerative disorders.     

Ataxia telangiectasia-mutated dependent DNA damage checkpoint functions regulate gene expression in human fibroblasts

The relationships between profiles of global gene expression and DNA damage checkpoint functions were studied in cells from patients with ataxia telangiectasia (AT). Three telomerase-expressing AT fibroblast lines displayed the expected hypersensitivity to ionizing radiation (IR) and defects in DNA damage checkpoints. Profiles of global gene expression in AT cells were determined at 2, 6, and 24 h after treatment with 1.5-Gy IR or sham treatment and were compared with those previously recognized in normal human fibroblasts. Under basal conditions, 160 genes or expressed sequence tags were differentially expressed in AT and normal fibroblasts, and these were associated by gene ontology with insulin-like growth factor binding and regulation of cell growth. On DNA damage, 1,091 gene mRNAs were changed in at least two of the three AT cell lines. When compared with the 1,811 genes changed in normal human fibroblasts after the same treatment, 715 were found in both AT and normal fibroblasts, including most genes categorized by gene ontology into cell cycle, cell growth, and DNA damage response pathways. However, the IR-induced changes in these 715 genes in AT cells usually were delayed or attenuated in comparison with normal cells. The reduced change in DNA damage response genes and the attenuated repression of cell cycle-regulated genes may account for the defects in cell cycle checkpoint function in AT cells.     

Ataxia telangiectasia mutated expression and activation in the testis

Ionizing radiation (IR) and consequent induction of DNA double-strand breaks (DSBs) causes activation of the protein ataxia telangiectasia mutated (ATM). Normally, ATM is present as inactive dimers; however, in response to DSBs, the ATM dimer partners cross-phosphorylate each other on serine 1981, and kinase active ATM monomers are subsequently released. We have studied the presence of both nonphosphorylated as well as active serine 1981 phosphorylated ATM (pS1981-ATM) in the mouse testis. In the nonirradiated testis, ATM was present in spermatogonia and spermatocytes until stage VII of the cycle of the seminiferous epithelium, whereas pS1981-ATM was found only to be present in the sex body of pachytene spermatocytes. In response to IR, ATM became activated by pS1981 cross-phosphorylation in spermatogonia and Sertoli cells. Despite the occurrence of endogenous programmed DSBs during the first meiotic prophase and the presence of ATM in both spermatogonia and spermatocytes, pS1981 phosphorylated ATM did not appear in spermatocytes after treatment with IR. These results show that spermatogonial ATM and ATM in the spermatocytes are differentially regulated. In the mitotically dividing spermatogonia, ATM is activated by cross-phosphorylation, whereas during meiosis nonphosphorylated ATM or differently phosphorylated ATM is already active. ATM has been shown to be present at the synapsed axes of the meiotic chromosomes, and in the ATM knock-out mice spermatogenesis stops at pachytene stage IV of the seminiferous epithelium, indicating that indeed nonphosphorylated ATM is functional during meiosis. Additionally, ATM is constitutively phosphorylated in the sex body where its continued presence remains an enigma.     

Disruption of klotho gene causes an abnormal energy homeostasis in mice

klotho mice, which genetically lack klotho gene expression, are characterized with various systemic phenotypes resembling human aging, and also with growth retardation. Here we show that klotho mice have a barely detectable amount of the white adipose tissue but their brown adipose tissue (BAT) is comparably preserved. Glucose tolerance and insulin sensitivity in klotho mice are increased compared to those in wild-type mice as revealed by intraperitoneal glucose and insulin tolerance tests. Uncoupling protein-1 gene expression of BAT and body temperature in klotho mice are lower than those in wild-type mice, suggesting that klotho mice have less energy expenditure than wild-type mice. Histological examination suggests that klotho mice possess less energy storage than wild-type mice with respect to glycogen in the liver and lipid in BAT. All these changes of parameters for energy homeostasis in klotho mice are very similar to those reported under food-restricted conditions. However, the amount of food intake is not different between klotho and wild-type mice when normalized for body weight. The present study elucidates the importance of klotho gene expression for the maintenance of normal energy homeostasis.     

Analyzing gene expression data in mice with the Neuro Behavior Ontology

We have applied the Neuro Behavior Ontology (NBO), an ontology for the annotation of behavioral gene functions and behavioral phenotypes, to the annotation of more than 1,000 genes in the mouse that are known to play a role in behavior. These annotations can be explored by researchers interested in genes involved in particular behaviors and used computationally to provide insights into the behavioral phenotypes resulting from differences in gene expression. We developed the OntoFUNC tool and have applied it to enrichment analyses over the NBO to provide high-level behavioral interpretations of gene expression datasets. The resulting increase in the number of gene annotations facilitates the identification of behavioral or neurologic processes by assisting the formulation of hypotheses about the relationships between gene, processes, and phenotypic manifestations resulting from behavioral observations.     

Characteristics of gene expression in morphologically abnormal needles of Scots pine

Expression of 373 genes in Scots pine morphologically abnormal needles has been analyzed with microarray analysis. It was found microarray technique allows detecting the minor changes in gene expression. The results obtained showed that changes in expression of 42 genes occur during the process of abnormal morphogenesis in the Pinus sylvestris needles. The increasing of expression level of genes encoding ubiquitin-conjugating enzyme and L-ascorbate peroxidase was revealed for all samples analyzed.     

Metabolic homeostasis in mice with disrupted Clock gene expression in peripheral tissues

The role of peripheral vs. central circadian rhythms and Clock in the maintenance of metabolic homeostasis and with aging was examined by using Clock(Delta19)+MEL mice. These have preserved suprachiasmatic nucleus and pineal gland rhythmicity but arrhythmic Clock gene expression in the liver and skeletal muscle. Clock(Delta19)+MEL mice showed fasting hypoglycemia in young-adult males, fasting hyperglycemia in older females, and substantially impaired glucose tolerance overall. Clock(Delta19)+MEL mice had substantially reduced plasma insulin and plasma insulin/glucose nocturnally in males and during a glucose tolerance test in females, suggesting impaired insulin secretion. Clock(Delta19)+MEL mice had reduced hepatic expression and loss of rhythmicity of gck, pfkfb3, and pepck mRNA, which is likely to impair glycolysis and gluconeogenesis. Clock(Delta19)+MEL mice also had reduced glut4 mRNA in skeletal muscle, and this may contribute to poor glucose tolerance. Whole body insulin tolerance was enhanced in Clock(Delta19)+MEL mice, however, suggesting enhanced insulin sensitivity. These responses occurred although the Clock(Delta19) mutation did not cause obesity and reduced plasma free fatty acids while increasing plasma adiponectin. These studies on clock-gene disruption in peripheral tissues and metabolic homeostasis provide compelling evidence of a relationship between circadian rhythms and the glucose/insulin and adipoinsular axes. It is, however, premature to declare that clock-gene disruption causes the full metabolic syndrome.     

Ataxia and paroxysmal dyskinesia in mice lacking axonally transported FGF14

Fibroblast growth factor 14 (FGF14) belongs to a distinct subclass of FGFs that is expressed in the developing and adult CNS. We disrupted the Fgf14 gene and introduced an Fgf14(N-beta-Gal) allele that abolished Fgf14 expression and generated a fusion protein (FGF14N-beta-gal) containing the first exon of FGF14 and beta-galactosidase. Fgf14-deficient mice were viable, fertile, and anatomically normal, but developed ataxia and a paroxysmal hyperkinetic movement disorder. Neuropharmacological studies showed that Fgf14-deficient mice have reduced responses to dopamine agonists. The paroxysmal hyperkinetic movement disorder phenocopies a form of dystonia, a disease often associated with dysfunction of the putamen. Strikingly, the FGF14N-beta-gal chimeric protein was efficiently transported into neuronal processes in the basal ganglia and cerebellum. Together, these studies identify a novel function for FGF14 in neuronal signaling and implicate FGF14 in axonal trafficking and synaptosomal function.     

正常与异常核型人类胚胎干细胞体外分化不同时期基因表达的异同

目的:研究正常核型和异常核型人胚胎干细胞(hESC)基因的表达异同.方法:实时荧光相对定量PCR检测两株正常核型(46,XX)及一株平衡易位13三体核型和一株三倍体核型hESC在体外自体分化不同时期X连锁基因PGK1、抑癌基因RBBP7及癌症基因GPC4的表达情况,并比较分化后不同时期、不同核型对父系印迹基因H19、IGF2R,母系印迹基因SNRPN及多能性调控基因OCT4、NANOG的影响.结果:随着分化时间的增加:①正常和异常核型hESC的PGK1均上调表达;②异常核型hESC抑癌基因RBBP7及癌症基因GPC4相对正常核型hESC呈现明显上调表达;③正常和异常核型hESC中印迹基因表达基本一致:H19、IGF2R上调而SNRPN表达变化不明显或下调;④多能性调控基因OCT4、NANOG在正常核型hESC中较在异常核型hESC中表达明显下降.结论:X连锁基因PGK1、印迹基因在hESC的发育过程中能维持正常调节而不受核型的影响.异常核型hESC抑癌基因、癌症基因在发育过程中的表达上调表明此种细胞具有更危险的发育前景,同时多能性基因在分化后仍能检出表明此种细胞分化能力较正常细胞弱.     

Altered behavioral rhythms and clock gene expression in mice with a targeted mutation in the Period1 gene

A group of specialized genes has been defined to govern the molecular mechanisms controlling the circadian clock in mammals. Their expression and the interactions among their products dictate circadian rhythmicity. Three genes homologous to Drosophila period exist in the mouse and are thought to be major players in the biological clock. Here we present the generation of mice in which the founding member of the family, Per1, has been inactivated by homologous recombination. These mice present rhythmicity in locomotor activity, but with a period almost 1 h shorter than wild-type littermates. Moreover, the expression of clock genes in peripheral tissues appears to be delayed in Per1 mutant animals. Importantly, light-induced phase shifting appears conserved. The oscillatory expression of clock genes and the induction of immediate-early genes in response to light in the master clock structure, the suprachiasmatic nucleus, are unaffected. Altogether, these data demonstrate that Per1 plays a distinct role within the Per family, as it may be involved predominantly in peripheral clocks and/or in the output pathways of the circadian clock.     

Defective epidermal growth factor gene expression in mice with polycystic kidney disease

The C57BL/6J-cpk mouse has an inheritable form of polycystic kidney disease similar to the autosomal recessive disorder seen in humans. Between approximately 1 and 3 weeks of age, affected cpk mice develop numerous large cysts in the collecting tubule segment of kidney nephrons. The present study examined the ontogeny of renal and submandibular gland prepro-epidermal growth factor (preproEGF) gene expression in the cpk mouse using Northern blot hybridization and immunohistochemistry. There was a virtual absence of renal preproEGF gene expression in cystic kidneys over the 3-week postnatal period, during which time renal preproEGF mRNA and proEGF/EGF protein normally reach significant levels. PreproEGF mRNA was expressed in salivary glands of cystic mice; however, this mRNA could not be further elevated with testosterone suggesting that there are abnormalities in the regulation of the preproEGF gene in the submandibular gland, as well as in the kidney. Since renal preproEGF expression during the early postnatal period occurs when collecting duct cysts form, it is possible that a deficiency in renal proEGF or EGF contributes to the rapid development of collecting duct cysts and the concomitant renal failure in the C57BL/6J-cpk cystic mouse.     

Clustered gene expression changes flank targeted gene loci in knockout mice

Background

Gene expression profiling using microarrays is a powerful technology widely used to study regulatory networks. Profiling of mRNA levels in mutant organisms has the potential to identify genes regulated by the mutated protein.

Methodology/Principle Findings

Using tissues from multiple lines of knockout mice we have examined genome-wide changes in gene expression. We report that a significant proportion of changed genes were found near the targeted gene.

Conclusions/Significance

The apparent clustering of these genes was explained by the presence of flanking DNA from the parental ES cell. We provide recommendations for the analysis and reporting of microarray data from knockout mice