Targeted suppression of E-cadherin gene expression in bovine preimplantation embryo by RNA interference technology using double-stranded RNA.
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.
Nganvongpanit
Korakot
K
Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, Endenicher Allee 15, Bonn, Germany.
Müller
Heike
H
Rings
Franca
F
Gilles
Markus
M
Jennen
Danyel
D
Hölker
Michael
M
Tholen
Ernst
E
Schellander
Karl
K
Tesfaye
Dawit
D
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
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