The Analysis of Differentially Expressed Gene by DNA Micro Array

DNA微阵列分析差异表达基因

• 批准号: 12671724
• 负责人: IWATA Fumino
• 金额: $ 2.24万
• 依托单位: Juntendo University, School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671724/
• 关键词: Macular degeneration; DNA array; Differential Gene Expression; ELOVL4; Cynomolgus; immunohistochemistry; AMD; ELO; cDNA; Subtraction

We have conducted histology, molecular biology, and genetic analysis of macular degeneration cynomolgus monkey with drusen. This monkey family previously discovered in National Primate Center in Tsukuba has been briefly characterized by observation of the retina and fluorescence imaging of the retina section. Sixty monkeys aged 15 to 24 years in propagation colonies were examined by fundus scope. Macular degeneration was observed in ten monkeys. Histological sections prepared from excised macular retina were stained by HE (hematoxylin and eosin), PAS (Periodic Acid Sciff), LFB (luxol fast blue) and MT (masson trichrome) and observed under light microscopy. Ultrastructure of drusen was examined by transmission electron microscopy. Hyper-pigmentation, partial vacuolation, increased autofluorescence, and PAS-positive granules in cell cytosol were observed in retinal pigment epithelium (RPE) of these affected monkey retina. However, minimum histological abnormality was observed in neural r … More etina and choroid. These observations indicate that lipofuscin granules are highly accumulated in RPE.. Molecular biology analysis using Clontech Inc, plastic DNA array with 8,000 genes were performed by labeling RNA extracted from affected and non-affected monkey retina by Cy3 and Cy5 respectively. After the normalization of all 8,000 genes, two samples were compared for differentially expressed gene. Fourteen genes were up-regulated by, 2.5 to 7.6-fold, while, five genes were down-regulated by 2.4 to 3.8-fold. Among the up-regulated genes, ATP synthase, H+ transporting, mitochondrial F0 complex, subunit f, isoform 2 gave more than 7.6-fold compare to normal. Lymphatic vessel endothelial hyaluronan receptor gave significant drop of 3.8-fold. Linkage analysis and candidate gene approach was performed in parallel to determine the gene responsible for the disease. The monkey homologue of the ELOVL4, gene responsible for autosomal dominant Stargardt-like macular dystrophy (STGD3) was cloned to determine any possible mutation in macular degeneration monkey. PCR of genomic DNA and 5'/3'-rapid amplification of cDNA ends were carried out to determine the complete sequence of the monkey ELOVL4 cDNA and the gene. ELOVL4 RNA was abundantly expressed in retina and thymus determined by real time quantitative PCR. Immunohistochemical analysis was performed using anti-human ELOVL4 peptide polyclonal antibody to localize ELOVL4 protein in monkey retina. No mutation was found in this gene. Less

我们对患有玻璃疣的黄斑变性食蟹猴进行了组织学、分子生物学和遗传学分析。这个猴子家族先前在筑波的国家灵长类动物中心发现，已经通过对视网膜的观察和视网膜切片的荧光成像进行了简要的表征。用眼底镜检查繁殖群中60只15 ~ 24岁的猕猴。在10只猴子中观察到黄斑变性。取黄斑区视网膜组织切片，分别进行HE、PAS、LFB和MT染色，光镜下观察。透射电镜观察玻璃疣的超微结构。在这些受累猴视网膜的视网膜色素上皮（RPE）中观察到色素沉着过度、部分空泡化、自体荧光增加和细胞胞质中PAS阳性颗粒。然而，在神经节中观察到最小的组织学异常， ...更多信息 视网膜和脉络膜。这些观察结果表明，脂褐质颗粒在RPE中高度积累。使用Clontech Inc，通过分别用Cy 3和Cy 5标记从患病和未患病的猴视网膜提取的RNA，进行具有8，000个基因的塑料DNA阵列的分子生物学分析。在所有8，000个基因的标准化之后，比较两个样品的差异表达基因。14个基因上调2.5 - 7.6倍，而5个基因下调2.4 - 3.8倍。其中ATP合成酶、H+转运蛋白、线粒体F0复合体、f亚基、亚型2等基因的表达量是正常的7.6倍以上。淋巴管内皮透明质酸受体下降3.8倍。连锁分析和候选基因的方法进行了平行，以确定基因负责的疾病。克隆了负责常染色体显性Stargardt样黄斑营养不良（STGD 3）的STGD VL 4基因的猴同源物，以确定黄斑变性猴中任何可能的突变。通过基因组DNA的PCR和cDNA末端的5 ′/3 ′快速扩增，确定了猴ERFVL 4 cDNA和基因的全序列。真实的实时定量PCR检测表明，HL-4 RNA在视网膜和胸腺中有丰富的表达。免疫组织化学分析使用抗人HPVL 4肽多克隆抗体进行，以定位HPVL 4蛋白在猴视网膜中。在该基因中未发现突变。少

项目成果

Iwata F, Reed GF, Caruso RC, Kuehl EM, Gall WA, Kaiser-Kuufer MI: "Correlation of visual acuity and ocular pigmentation with the 16-bp duplication in the HPS-1 gene of Hermansky-Pudlak syndrome, a form of albinism"Ophthalmology. 107. 783-789 (2000)

Iwata F、Reed GF、Caruso RC、Kuehl EM、Gall WA、Kaiser-Kuufer MI：“视力和眼部色素沉着与 Hermansky-Pudlak 综合征（一种白化病）HPS-1 基因中 16 bp 重复的相关性

Iwata F, Reed GF, Caruso RC, Kuehl EM, Gahl WA, Kaiser-Kupfer Ml: "Correlation of visual acuity and ocular pigmentation with the 16-bp duplication in the HPS-1 gene of Hermansky-Pudlak syndrome, a form of albinism."Ophthalmology. 107. 783-9 (2000)

Iwata F、Reed GF、Caruso RC、Kuehl EM、Gahl WA、Kaiser-Kupfer Ml：“视力和眼部色素沉着与 Hermansky-Pudlak 综合征（一种白化病）HPS-1 基因中 16 bp 重复的相关性

Jiao X, Munier FL, Iwata F, Hayakawa M, Kanai A, Lee J, Schorderet DF, Chen MS, Kaiser-Kupfer M, Hejtmancik JF.: "Genetic linkage of Bietti crystallin corneoretinal dystrophy to chromosome 4q35"Am J Hum Genet.. 67. 1309-1313 (2000)

Jiao X、Munier FL、Iwata F、Hayakawa M、Kanai A、Lee J、Schorderet DF、Chen MS、Kaiser-Kupfer M、Hejtmancik JF.：“Bietti 晶状体角膜视网膜营养不良与染色体 4q35 的遗传连锁”Am J Hum Genet。

Gahl WA, Kuehl EM, Iwata F, Lindblad A, Kaiser-Kupfer MI: "Corneal crystals in nephropathic cystinosis natural history and treatment. with cysteamine eyedrops"Mol Genet Metab. 71. 100-120 (2000)

Gahl WA、Kuehl EM、Iwata F、Lindblad A、Kaiser-Kupfer MI：“肾病性胱氨酸病自然史和治疗中的角膜晶体。使用半胱胺滴眼液”Mol Genet Metab。

Gahl WA, Kuehl EM, Iwata F, Landblad A, Kaiser-Kupfer Ml.: "Corneal crystals in nephropathic cystinosis : natural history and treatment with cysteamine eyedrops."Mol. Genet. Metab. 71. 100-20 (2000)

Gahl WA、Kuehl EM、Iwata F、Landblad A、Kaiser-Kupfer Ml.：“肾病性胱氨酸病的角膜晶体：自然史和半胱胺滴眼液治疗。”Mol。