RNA interference as a tool to study gene function in bovine oocytes.
RNA interference (RNAi) has become a well-established technique to study gene function in several species. Our objective was to develop a RNAi approach to study gene function in bovine oocytes. In the first experiment, three different treatments including a 20 min exposure to cytochalasin B, a 6 hr maturation in cycloheximide, and a combination of these two treatments were tested to improve oocyte survival following microinjection. The cycloheximide/cytochalasin B treatment greatly increased (P<0.02) the survival rate of the microinjected oocytes. In the second experiment, we assessed the effect of both cyclin B1 and GFP dsRNA on cyclin B1 mRNA and protein expression. The injection of cyclin B1 dsRNA resulted in a decrease in cyclin B1 mRNA and protein, while the cyclin B2 mRNA remained unaffected. Furthermore, the injection of GFP dsRNA did not interfere with cyclin B1 mRNA or protein nor with the ability of the oocyte to mature properly. In addition, the lack of cyclin B1 in the oocyte led to activation in 10% of the oocytes as evidenced by the presence of a pronucleus. However, the use of an additional 10 hr of maturation in the presence of 6-dimethylaminopurine (6-DMAP) prevented germinal vesicle breakdown and allowed a longer exposure to dsRNA. This procedure increased the percentage of activated oocytes to 33% and is likely to result from an increased length of time for dsRNA processing and for degradation of the cyclin B1 mRNA to occur. In conclusion, RNAi represents a useful technique to study gene function in the bovine oocyte.
Paradis
François
F
Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4.
Vigneault
Christian
C
Robert
Claude
C
Sirard
Marc-André
MA
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D019926:Cyclin B / Q000235:genetics / Q000378:metabolism; D056744:Cyclin B1; D003513:Cycloheximide / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D005260:Female; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D008845:Microinjections; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000494:pharmacology
D019926:Cyclin B; D056744:Cyclin B1; D047428:Protein Kinase Inhibitors; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D003571:Cytochalasin B; C001020:N(6),N(6)-dimethyladenine; D003513:Cycloheximide; D000225:Adenine