Injection of mammalian metaphase II oocytes with short interfering RNAs to dissect meiotic and early mitotic events.
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.
Amanai
Manami
M
Laboratory of Mammalian Molecular Embryology, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.
Shoji
Shisako
S
Yoshida
Naoko
N
Brahmajosyula
Manjula
M
Perry
Anthony C F
AC
D000818:Animals; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D047108:Embryonic Development / Q000502:physiology; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D005306:Fertilization / Q000502:physiology; D008540:Meiosis / Q000502:physiology*; D008677:Metaphase; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008845:Microinjections; D008938:Mitosis / Q000502:physiology*; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology* / Q000502:physiology*; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000493:pharmacokinetics*
D064199:Cdc20 Proteins; C577491:Cdc20 protein, mouse; D018797:Cell Cycle Proteins; C498959:Emi1 protein, mouse; C509434:Emi2 protein, mouse; D044783:F-Box Proteins; D011506:Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering