Microinjection of plasmids into meiotically incompetent 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 mammalian double-stranded RNA (dsRNA) expression vector into meiotically incompetent mouse oocytes by microinjection. During mouse oocyte growth, there is a progressive decline in transcriptional activity, such that the fully-grown oocyte is transcriptionally quiescent. For this reason, the microinjection of plasmids should be performed in growing oocytes, obtained from 12- to 13-d-old females. Microinjected oocytes can be cultured for up to 2 d. 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; D005260:Female; D005822:Genetic Vectors; D008540:Meiosis; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections / Q000379:methods*; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019076:Transgenes
D012330:RNA, Double-Stranded