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1 December 2006 Injection of Mammalian Metaphase II Oocytes with Short Interfering RNAs to Dissect Meiotic and Early Mitotic Events
Manami Amanai, Shisako Shoji, Naoko Yoshida, Manjula Brahmajosyula, Anthony C. F. Perry
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Abstract

The manipulation of mammalian metaphase II (mII) oocytes has illuminated the mechanisms of fertilization and early embryogenesis and is central to nuclear transfer. Although RNA interference (RNAi) would greatly facilitate this type of manipulation, its application to mature, developmentally competent mII oocytes has not been evaluated. We report efficient RNAi by the injection of short interfering RNAs (siRNAs) into mII oocytes. The levels of the target mRNA and corresponding protein were rapidly and efficiently reduced. The siRNAs were effective when injected in the subnanomolar to nanomolar range and induced concurrently RNAi of multiple targets, revealing the kinetic parameters of RNAi in mII oocytes. Coinjection of sperm with siRNA functionally abolished the transcripts in the resultant blastocysts and in cloned embryos into which siRNA was coinjected during somatic cell nuclear transfer. The RNAi method was used to dissect the early mitotic roles of meiotic regulators, which suggests that CDC20 is essential for the first mitotic division, while EMI1 and EMI2 are not essential for this process. Our results show that siRNA injection of oocytes confers temporal control of RNAi in the analysis and manipulation of key processes in mammalian meiosis and early embryogenesis.

Manami Amanai, Shisako Shoji, Naoko Yoshida, Manjula Brahmajosyula, and Anthony C. F. Perry "Injection of Mammalian Metaphase II Oocytes with Short Interfering RNAs to Dissect Meiotic and Early Mitotic Events," Biology of Reproduction 75(6), 891-898, (1 December 2006). https://doi.org/10.1095/biolreprod.106.054213
Received: 27 May 2006; Accepted: 1 August 2006; Published: 1 December 2006
JOURNAL ARTICLE
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KEYWORDS
early development
embryo
fertilization
gamete biology
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