title: Multiple renal cyst development but not situs abnormalities in transgenic RNAi mice against Inv::GFP rescue gene.
abstract: In this study we generated RNA interference (RNAi)-mediated gene knockdown transgenic mice (transgenic RNAi mice) against the functional Inv gene. Inv mutant mice show consistently reversed internal organs (situs inversus), multiple renal cysts and neonatal lethality. The Inv::GFP-rescue mice, which introduced the Inv::GFP fusion gene, can rescue inv mutant mice phenotypes. This indicates that the Inv::GFP gene is functional in vivo. To analyze the physiological functions of the Inv gene, and to demonstrate the availability of transgenic RNAi mice, we introduced a short hairpin RNA expression vector against GFP mRNA into Inv::GFP-rescue mice and analyzed the gene silencing effects and Inv functions by examining phenotypes. Transgenic RNAi mice with the Inv::GFP-rescue gene (Inv-KD mice) down-regulated Inv::GFP fusion protein and showed hypomorphic phenotypes of inv mutant mice, such as renal cyst development, but not situs abnormalities or postnatal lethality. This indicates that shRNAi-mediated gene silencing systems that target the tag sequence of the fusion gene work properly in vivo, and suggests that a relatively high level of Inv protein is required for kidney development in contrast to left/right axis determination. Inv::GFP protein was significantly down-regulated in the germ cells of Inv-KD mice testis compared with somatic cells, suggesting the existence of a testicular germ cell-specific enhanced RNAi system that regulates germ cell development. The Inv-KD mouse is useful for studying Inv gene functions in adult tissue that are unable to be analyzed in inv mutant mice showing postnatal lethality. In addition, the shRNA-based gene silencing system against the tag sequence of the fusion gene can be utilized as a new technique to regulate gene expression in either in vitro or in vivo experiments.
authors.affiliation: Laboratory of Molecular Embryology, Department of Bioscience, Kitasato University School of Science, Sagamihara, Kanagawa, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kamijho
authors.affiliation: Laboratory of Molecular Embryology, Department of Bioscience, Kitasato University School of Science, Sagamihara, Kanagawa, Japan.
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Shiozaki
authors.affiliation: Laboratory of Molecular Embryology, Department of Bioscience, Kitasato University School of Science, Sagamihara, Kanagawa, Japan.
authors.forename: Eiki
authors.initials: E
authors.lastname: Sakurai
authors.affiliation: Laboratory of Molecular Embryology, Department of Bioscience, Kitasato University School of Science, Sagamihara, Kanagawa, Japan.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Hanaoka
authors.affiliation: Laboratory of Molecular Embryology, Department of Bioscience, Kitasato University School of Science, Sagamihara, Kanagawa, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Watanabe
mesh_terms: D000375:Aging; D000818:Animals; D050939:Gene Fusion; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007668:Kidney / Q000196:embryology; D052177:Kidney Diseases, Cystic / Q000235:genetics*; D008297:Male; D008822:Mice, Transgenic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D012857:Situs Inversus / Q000235:genetics; D013737:Testis / Q000166:cytology / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics*
chemical_list: C491947:Invs protein, mouse; D034741:RNA, Small Interfering; D014157:Transcription Factors; D049452:Green Fluorescent Proteins