title: cDNA cloning of porcine PKD2 gene and RNA interference in LLC-PK1 cells.
abstract: 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.
authors.affiliation: Tianjin Research Centre of Basic Medical Science, Tianjin Medical University, Qixiangtai Road, Heping District, Tianjin, 300070, China.
authors.forename: Qingsong
authors.initials: Q
authors.lastname: Wang
authors.forename: Haifang
authors.initials: H
authors.lastname: Yin
authors.forename: Jin
authors.initials: J
authors.lastname: He
authors.forename: Jianhua
authors.initials: J
authors.lastname: Ye
authors.forename: Fangrong
authors.initials: F
authors.lastname: Ding
authors.forename: Shaohua
authors.initials: S
authors.lastname: Wang
authors.forename: Xiaoxiang
authors.initials: X
authors.lastname: Hu
authors.forename: Qingyong
authors.initials: Q
authors.lastname: Meng
authors.forename: Ning
authors.initials: N
authors.lastname: Li
mesh_terms: D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D004195:Disease Models, Animal; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism; D018374:LLC-PK1 Cells; D016891:Polycystic Kidney, Autosomal Dominant / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D013552:Swine; D050396:TRPP Cation Channels / Q000235:genetics* / Q000378:metabolism; D014018:Tissue Distribution
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D034741:RNA, Small Interfering; D050396:TRPP Cation Channels