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Type of publication: Journal Article
Type of document: Full Paper

Year: 2006

Authors: Nganvongpanit, K; Müller, H; Rings, F; Hoelker, M; Jennen, D; Tholen, E; Havlicek, V; Besenfelder, U; Schellander, K; Tesfaye, D

Title: Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA.

Source: Reproduction. 2006; 131(5):861-874

Authors Vetmeduni Vienna:

Besenfelder Urban
Havlicek Vitezslav

Vetmed Research Units
Department for Agrobiotechnology (IFA Tulln)
Institute of Animal Breeding and Genetics

RNA interference (RNAi) has been used for selective degradation of an mRNA transcript or inhibiting its translation to a functional protein in various species. Here, we applied the RNAi approach to suppress the expression of the maternal transcript C-mos and embryonic transcripts Oct-4 in bovine oocytes and embryos respectively, using microinjection of sequence-specific double-stranded RNA (dsRNA). For this, 435 bp C-mos and 341 bp Oct-4 dsRNA were synthesized and microinjected into the cytoplasm of immature oocytes and zygotes respectively. In experiment 1, immature oocytes were categorized into three groups: those injected with C-mos dsRNA, RNase-free water and uninjected controls. In experiment 2, in vitro produced zygotes were categorized into three groups: those injected with Oct-4 dsRNA, RNase-free water and uninjected controls. The developmental phenotypes, the level of mRNA and protein expression were investigated after treatment in both experiments. Microinjection of C-mos dsRNA has resulted in 70% reduction of C-mos transcript after maturation compared to the water-injected and uninjected controls (P<0.01). Microinjection of zygotes with Oct-4 dsRNA has resulted in 72% reduction in transcript abundance at the blastocyst stage compared to the uninjected control zygotes (P<0.01). Moreover, a significant reduction in the number of inner cell mass (ICM) cells was observed in Oct-4 dsRNA-injected embryos compared to the other groups. From oocytes injected with C-mos dsRNA, 60% showed the extrusion of the first polar body compared to 50% in water-injected and 44% in uninjected controls. Moreover, only oocytes injected with C-mos dsRNA showed spontaneous activation. In conclusion, our results demonstrated that sequence-specific dsRNA can be used to knockdown maternal or embryonic transcripts in bovine embryogenesis.

Keywords Pubmed: Animals
Base Sequence
Blotting, Western/methods
Cells, Cultured
DNA Primers/genetics
Embryonic Development
Fertilization in Vitro
Gene Expression Regulation, Developmental*
Molecular Sequence Data
Octamer Transcription Factor-3/analysis
Octamer Transcription Factor-3/genetics*
Proto-Oncogene Proteins c-mos/analysis
Proto-Oncogene Proteins c-mos/genetics*
RNA Interference*
RNA, Double-Stranded/administration & dosage*
Reverse Transcriptase Polymerase Chain Reaction
Staining and Labeling
Transcription, Genetic

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