title: RNAi-mediated knockdown of mouse melanocortin-4 receptor in vitro and in vivo, using an siRNA expression construct based on the mir-187 precursor.
abstract: RNA interference (RNAi) is a powerful tool for the study of gene function in mammalian systems, including transgenic mice. Here, we report a gene knockdown system based on the human mir-187 precursor. We introduced small interfering RNA (siRNA) sequences against the mouse melanocortin-4 receptor (mMc4r) to alter the targeting of miR-187. The siRNA-expressing cassette was placed under the control of the cytomegalovirus (CMV) early enhancer/chicken β-actin promoter. In vitro, the construct efficiently knocked down the gene expression of a co-transfected mMc4r-expression vector in cultured mammalian cells. Using this construct, we generated a transgenic mouse line which exhibited partial but significant knockdown of mMc4r mRNA in various brain regions. Northern blot analysis detected transgenic expression of mMc4r siRNA in these regions. Furthermore, the transgenic mice fed a normal diet ate 9% more and were 30% heavier than wild-type sibs. They also developed hyperinsulinemia and fatty liver as do mMc4r knockout mice. We determined that this siRNA expression construct based on mir-187 is a practical and useful tool for gene functional studies in vitro as well as in vivo.
authors.affiliation: Research Unit/Neuroscience, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida, Aoba-ku, Yokohama, Kanagawa 227-0033, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Kato
authors.forename: Yi-Ying
authors.initials: YY
authors.lastname: Huang
authors.forename: Mina
authors.initials: M
authors.lastname: Matsuo
authors.forename: Yoko
authors.initials: Y
authors.lastname: Takashina
authors.forename: Kazuyo
authors.initials: K
authors.lastname: Sasaki
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Horai
authors.forename: Aya
authors.initials: A
authors.lastname: Juni
authors.forename: Shin-Ichi
authors.initials: SI
authors.lastname: Kamijo
authors.forename: Kaoru
authors.initials: K
authors.lastname: Saigo
authors.forename: Kumiko
authors.initials: K
authors.lastname: Ui-Tei
authors.forename: Hajime
authors.initials: H
authors.lastname: Tei
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D002645:Chickens / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D035683:MicroRNAs / Q000235:genetics; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012319:RNA Polymerase II / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D044105:Receptor, Melanocortin, Type 4 / Q000235:genetics* / Q000378:metabolism
chemical_list: D000199:Actins; C574273:MC4R protein, mouse; D035683:MicroRNAs; D034741:RNA, Small Interfering; D044105:Receptor, Melanocortin, Type 4; D012319:RNA Polymerase II