cDNA cloning of porcine PKD2 gene and RNA interference in LLC–PK1 cells Qingsong Wang a,b , Haifang Yin a , Jin He b , Jianhua Ye b , Fangrong Ding b , Shaohua Wang b , Xiaoxiang Hu b , Qingyong Meng b , Ning Li b, ⁎ a Tianjin Research Centre of Basic Medical Science, Tianjin Medical University, Qixiangtai Road, Heping District, Tianjin, 300070, China b State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100193, China abstract article info Article history: Accepted 24 January 2011 Available online 28 January 2011 Received by M. Schartl Keywords: ADPKD TRPP2 Polycystin-2 Knockdown Cell proliferation 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. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Autosomal dominant polycystic kidney (ADPKD) is a common genetic disease, affecting up to 1 in 400 people worldwide. In ADPKD (Kaariainen, 1987) kidneys are typically enlarged with numerous cysts of varying size, which are randomly distributed throughout the renal parenchyma and arise from any nephron segment (Gabow, 1993). The cysts show thickened basement membranes with surrounding interstitial fibrosis, though their epithelia maintain an active capacity for fluid secretion and re-absorption (Chapman et al., 2003; Grantham, 2006). End-stage renal disease (ESRD) occurs in the majority of ADPKD cases (Torres et al., 2007), with approximately 85% of ADPKD cases caused by mutations in PKD1 (Reeders et al., 1985; Pignatelli et al., 1992) and PKD2 mutations account for the remaining 15% (Bachner et al., 1990; Kimberling et al., 1993; Mochizuki et al., 1996). A third locus PKD3 (Daoust et al., 1995), which is likely responsible for a small percentage of ADPKD cases, remains to be characterized. Currently there is no effective therapeutic intervention available for ADPKD. In order to define the etiology of and develop effective treatments for ADPKD, a number of animal models have been investigated; however most of them could not recapitulate the disease progression and symptoms observed in ADPKD patients due to either the lethality in homozygotes or milder phenotypes in heterozygotes. Therefore it is imperative to develop an animal model which can mimic the ADPKD patients more closely. Swine is commonly regarded as the ideal animal disease model due to its significant similarity to humans physiologically and anatomically. Therefore, the identification and characterization of the porcine PKD2 gene has the potential to provide information critical to the establishment of an appropriate animal model and subsequent advances in the development of therapeutic treatment for ADPKD. It is known that the human PKD2 gene and its protein – transient receptor potential polycystin-2 (TRPP2) – belong to the TRP channel family. In this family, there are six transmembrane domains plus one N-terminal and one C-terminal intracellular domains (Mochizuki et al., 1996). It functions as a calcium-permeable cation channel that mediates calcium fluxes across plasma membrane (Chen et al., 2008), Gene 476 (2011) 38–45 Abbreviations: ADPKD, autosomal dominant polycystic kidney; ESRD, end-stage renal disease; RNAi, RNA interference; siRNA, small interfering RNA; shRNA, short hairpin RNA; ER, endoplasmic reticulum; PI3R, type I inositol 1,4,5-triphosphate receptor; EST, Expressed Sequence Tags; RACE, Rapid Amplification of cDNA Ends; PI, propidium iodide; PERK, pancreatic ER-resident eIF2 kinase; TRPP, transient receptor potential polycystic. ⁎ Corresponding author. Fax: +86 10 6273 3904. E-mail address: ninglcau@cau.edu.cn (N. Li). 0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.01.017 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene