title: Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro.
abstract: Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro.
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, 241-438 Shimoshinjho Nakano, Akita 010-0195, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, 241-438 Shimoshinjho Nakano, Akita 010-0195, Japan.
authors.forename: Shiori
authors.initials: S
authors.lastname: Sato
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, 241-438 Shimoshinjho Nakano, Akita 010-0195, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kikuchi
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, 241-438 Shimoshinjho Nakano, Akita 010-0195, Japan.
authors.forename: Asumi
authors.initials: A
authors.lastname: Nonaka
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, 241-438 Shimoshinjho Nakano, Akita 010-0195, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kumagai
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, 241-438 Shimoshinjho Nakano, Akita 010-0195, Japan; Akita Research Institute of Food and Brewing, 4-26 Sanuki, Araya-machi, Akita 010-1623, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Sasaki
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, 241-438 Shimoshinjho Nakano, Akita 010-0195, Japan. Electronic address: makoba@akita-pu.ac.jp.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Kobayashi
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000235:genetics; D055785:Gene Knockdown Techniques / Q000379:methods*; D051379:Mice; D060172:Morpholinos / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis / Q000235:genetics
chemical_list: D060172:Morpholinos; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse
keywords: Antisense; Endo-Porter; Knockdown; Morpholino oligo; OCT4; Preimplantation mouse embryo