cDNA cloning of porcine PKD2 gene and RNA interference in LLC-PK1 cells
cDNA cloning of porcine PKD2 gene and RNA interference in LLC-PK1 cells
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猪PKD2基因的cDNA克隆及LLC-PK1细胞中的RNA干扰
DOI：
10.1016/j.gene.2011.01.017
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发表时间：
2011-05-01
期刊：
影响因子：
3.5
通讯作者：
Li, Ning
中科院分区：
文献类型：
--
作者：
Wang, Qingsong;Yin, Haifang;Li, Ning
PKD 2基因突变导致常染色体显性多囊肾病（ADPKD），这是一种常见的遗传性疾病，经常导致终末期肾病（ESRD）。猪在生理学和解剖学上与人类具有很大的相似性，因此是一个很好的模型系统，可以用来破译PKD 2基因的结构和功能，并鉴定潜在的治疗靶点。在这里，我们报告的猪PKD 2 cDNA的克隆和表征表明，全长基因（3370个碱基）在肾脏中高度表达，在肝脏中的表达最小。RNA干扰（RNAi）是一种很有前途的工具，使识别的必要组成部分，利用参与细胞过程的途径。因此，我们设计了4个shRNAs和9个siRNA，靶向猪PKD 2基因外显子3至9的区域，该区域被认为是导致ADPKD严重程度的关键区域。在HeLa细胞和猪肾细胞（LLC-PK 1）中的结果表明，sh 12可以有效地敲除PKD 2基因，敲除效率为51%，P1和P2是最有效的siRNA，与未处理的对照相比，分别抑制PKD 2基因表达的85%和77%。随后对瞬时受体电位多囊（TRPP）2通道蛋白的功能研究表明，siRNA P1和P2诱导的TRPP 2表达的降低可以解除细胞周期从G 0/G1期的阻滞，促进细胞向S和G2期的进展。因此，我们的数据提供了PKD 2基因中潜在敲低靶位点的证据，并为未来一代转基因ADPKD敲低动物模型铺平了道路。(C)2011 Elsevier B. V.保留所有权利。
Mutations in the PKD2 gene cause autosomal dominant polycystic kidney disease (ADPKD), a common, inherited disease that frequently leads to end-stage renal disease (ESRD). Swine show substantial similarity to humans physiologically and anatomically, and are therefore a good model system in which to decipher the structure and function of the PKD2 gene and to identify potential therapeutic targets. Here we report the cloning and characterization of the porcine PKD2 cDNA showing that the full-length gene (3370 bases) is highly expressed in kidney, with minimal expression in the liver. RNA interference (RNAi) is a promising tool to enable identification of the essential components necessary for exploitation of the pathway involved in cellular processes. We therefore designed four shRNAs and nine siRNAs targeting the region of the porcine PKD2 gene from exons 3 to 9, which is supposed to be a critical region contributing to the severity of ADPKD. The results from HeLa cells with the dual-luciferase reporter system and porcine kidney cells (LLC-PK1) showed that sh12 could efficiently knock down the PKD2 gene with an efficiency of 51% and P1 and P2 were the most effective siRNAs inhibiting 85% and 77% respectively of PKD2 expression compared with untreated controls. A subsequent functional study of the transient receptor potential polycystic (TRPP) 2 channel protein indicated that the decreased expression of TRPP2 induced by siRNA P1 and P2 could release the arrest of the cell cycle from G0/G1 promoting progression to S and G2 phases. Our data, therefore, provides evidence of potential knock-down target sites in the PKD2 gene and paves the way for the future generation of transgenic ADPKD knock-down animal models. (C) 2011 Elsevier B.V. All rights reserved.