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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


Abstract:
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
Blastocyst/cytology
Blotting, Western/methods
Cattle
Cells, Cultured
DNA Primers/genetics
Embryonic Development
Female
Fertilization in Vitro
Gene Expression Regulation, Developmental*
Microinjections
Molecular Sequence Data
Octamer Transcription Factor-3/analysis
Octamer Transcription Factor-3/genetics*
Oogenesis
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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