Identification and localization of novel genes preferentially expressed in human kidney glomerulus
Identification and localization of novel genes preferentially expressed in human kidney glomerulus
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DOI：
10.1111/j.1440-1797.2008.01009.x
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发表时间：
2009-02-01
期刊：
影响因子：
2.5
通讯作者：
Yamamoto, Tadashi
中科院分区：
文献类型：
--
作者：
Cuellar, Lino Munoz;Fujinaka, Hidehiko;Yamamoto, Tadashi
为了寻找肾小球中大量表达的新基因，我们构建了肾小球cDNA文库，并通过与斯坦福大学微阵列数据库（SMD）的比对、逆转录聚合酶链反应（RT-PCR）和原位杂交（ISH）验证了该文库的可靠性。在来自文库的5 × 103个克隆中，91个三个以上克隆的UniGene簇被鉴定为“肾小球丰富基因”。所有这些基因都被称为SMD，其中18个基因被定义为“肾小球优先基因”。选择四个未知基因--IFI 27、CRHBP、FLJ 10154和SEMA 5 B--进行RT-PCR以比较肾小球与皮质和髓质中的表达，并进行ISH以检查肾小球定位。此外，选择肾小球丰富但未在SMD中列出的三个未知基因-DDX 5、HSPC 138和MGC 10940-用于RT-PCR和ISH。最后，对新月体肾炎肾活检标本进行原位杂交，检测肾小球CRHBP mRNA的表达情况，从筛选出的7个肾小球丰富基因中，有6个经RT-PCR证实为“肾小球优先基因”。原位杂交结果显示，所有这些基因均在足细胞中表达。CRHBP mRNA在单个足细胞中的表达在正常和新月体肾小球中没有变化，首次证实了这些新基因在肾小球中的优先表达和足细胞定位。由于这些基因中有一些未被SMD收录，我们的数据库可以成为一个有用的工具，以寻找新的人类肾小球基因。
To find novel genes abundantly and preferentially expressed in human glomerulus, we constructed a glomerular cDNA library and verified the reliability of our database by comparison with the Stanford Microarray Database (SMD), followed by reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization (ISH).RNA was extracted from normal human glomeruli, and the cDNA library was constructed by plasmid cloning. Out of 5 x 10(3) clones from the library, 91 UniGene clusters of more than three clones were identified as 'glomerular-abundant genes'. All these genes were referred to the SMD, and 18 genes were defined as 'glomerular preferential genes'. Four unknown genes - IFI27, CRHBP, FLJ10154 and SEMA5B - were selected for RT-PCR to compare expression in the glomerulus with that in the cortex and medulla, and for ISH to examine glomerular localization. Also, three unknown genes that were glomerular abundant but not listed in the SMD - DDX5, HSPC138, and MGC10940 - were selected for RT-PCR and ISH. Finally, a kidney biopsy specimen of crescentic glomerulonephritis was used for ISH to examine glomerular expression for CRHBP mRNA.Among the selected seven glomerular-abundant genes, six were confirmed as 'glomerular preferential genes' by RT-PCR. By ISH, all these genes were demonstrated in podocytes. The expression of CRHBP mRNA in a single living podocyte was not changed between normal and crescentic glomerulus.Glomerular preferential expression and podocyte localization of these novel genes have been demonstrated for the first time. Because some of these genes were not listed in SMD, our database can be a useful tool to find novel human glomerular genes.