title: Targeted suppression of E-cadherin gene expression in bovine preimplantation embryo by RNA interference technology using double-stranded RNA.
abstract: RNA interference (RNAi) has become acknowledged as an effective and useful tool to study gene function in diverse groups of cells. We aimed to suppress the expression of the E-cadherin gene during in vitro development of bovine preimplantation embryos using RNAi approach. In this experiment the effect of microinjection of E-cadherin and Oct-4 (as control) double-stranded (ds) RNA on the mRNA and protein expression level of the target E-cadherin gene was investigated. For this, a 496 bp long bovine E-cadherin and 341 bp long Oct-4 dsRNA sample were prepared using in vitro transcription. In vitro produced bovine zygotes were categorized into four treatment groups including those injected with E-cadherin dsRNA, Oct-4 dsRNA, RNase-free water, and uninjected controls. While the injection of E-cadherin dsRNA resulted in the reduction of E-cadherin mRNA and protein levels at the morula and blastocyst stage, the transcript and protein product remained unaffected in the Oct-4 dsRNA, water injected and uninjected control groups. The relative abundance of E-cadherin mRNA in the E-cadherin dsRNA injected morula stage embryos was reduced by 80% compared to the control group (P < 0.05). The Western blot analysis also showed a significant decrease in the E-cadherin protein (119 kDa) in E-cadherin dsRNA injected embryos compared to the other three groups. Microinjection of E-cadherin dsRNA has resulted only 22% blastocyst rate compared to 38%-40% in water injected and uninjected controls. In conclusion, our results indicated the suppression of E-cadherin mRNA and protein has resulted in lower blastocyst rate and the RNAi technology is a promising approach to study the function of genes in early bovine embryogenesis.
authors.affiliation: Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, Endenicher Allee 15, Bonn, Germany.
authors.forename: Korakot
authors.initials: K
authors.lastname: Nganvongpanit
authors.forename: Heike
authors.initials: H
authors.lastname: Müller
authors.forename: Franca
authors.initials: F
authors.lastname: Rings
authors.forename: Markus
authors.initials: M
authors.lastname: Gilles
authors.forename: Danyel
authors.initials: D
authors.lastname: Jennen
authors.forename: Michael
authors.initials: M
authors.lastname: Hölker
authors.forename: Ernst
authors.initials: E
authors.lastname: Tholen
authors.forename: Karl
authors.initials: K
authors.lastname: Schellander
authors.forename: Dawit
authors.initials: D
authors.lastname: Tesfaye
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000187:drug effects* / Q000502:physiology; D015820:Cadherins / Q000187:drug effects / Q000235:genetics*; D002417:Cattle; D051187:Desmocollins; D005307:Fertilization in Vitro / Q000379:methods; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000235:genetics; D008969:Molecular Sequence Data; D050814:Octamer Transcription Factor-3 / Q000187:drug effects / Q000235:genetics; D010641:Phenotype; D034622:RNA Interference / Q000502:physiology*; D012330:RNA, Double-Stranded / Q000494:pharmacology*; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics; D014158:Transcription, Genetic; D051176:beta Catenin / Q000187:drug effects / Q000235:genetics
chemical_list: D015820:Cadherins; C081561:DSC2 protein, human; D051187:Desmocollins; D008562:Membrane Glycoproteins; D050814:Octamer Transcription Factor-3; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D051176:beta Catenin