title: Characterization of zebrafish polymerase III promoters for the expression of short-hairpin RNA interference molecules.
abstract: RNA interference (RNAi) is a powerful, sequence specific, and long-lasting method of gene knockdown, and can be elicited by the expression of short-hairpin RNA (shRNA) molecules driven via polymerase III type 3 promoters from a DNA vector or transgene. To further develop RNAi as a tool in zebrafish, we have characterized the zebrafish U6 and H1 snRNA promoters and compared the efficiency of each of the promoters to express an shRNA and silence a reporter gene, relative to previously characterized U6 promoters from pufferfish, chicken, and mouse. Our results show that the zebrafish polymerase III promoters were capable of effective gene silencing in the zebrafish ZF4 cell line, but were ineffective in mammalian Vero cells. In contrast, mouse and chicken promoters were active in Vero but not ZF4 cells, highlighting the importance of homologous promoters to achieve effective silencing.
authors.affiliation: 1 CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong , Victoria, Australia .
authors.forename: Brian D
authors.initials: BD
authors.lastname: Clarke
authors.forename: David M
authors.initials: DM
authors.lastname: Cummins
authors.forename: Ken A
authors.initials: KA
authors.lastname: McColl
authors.forename: Alister C
authors.initials: AC
authors.lastname: Ward
authors.forename: Tim J
authors.initials: TJ
authors.lastname: Doran
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D012320:RNA Polymerase III / Q000235:genetics*; D034741:RNA, Small Interfering / Q000378:metabolism*; D014709:Vero Cells; D015027:Zebrafish / Q000235:genetics*
chemical_list: D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012320:RNA Polymerase III