Microinjection of dsRNA into fully-grown mouse oocytes.
RNA interference (RNAi) is a suitable method for sequence-specific post-transcriptional gene silencing in a number of model systems. The following protocol describes delivery of a double-stranded RNA (dsRNA) of choice into fully-grown, germinal vesicle-intact (GV) mouse oocytes by microinjection. Microinjected oocytes can be cultured for up to 2 d or they can be matured to metaphase II. The metaphase II eggs can be fertilized in vitro and cultured up to the blastocyst stage. The efficiency of knockdown by RNAi can be assayed by quantitative reverse transcriptase (RT)-PCR (qPCR).
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA. steinpau@sas.upenn.edu
Paula
P
Stein
D000818:Animals; D003470:Culture Media / Q000378:metabolism; D005260:Female; D008677:Metaphase; D051379:Mice; D008845:Microinjections / Q000379:methods*; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism; D010641:Phenotype; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D003470:Culture Media; D012330:RNA, Double-Stranded; D012333:RNA, Messenger