RNA interference as a tool to study gene function in bovine oocytes

Abstract

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.

Exploring the function of long non-coding RNA in the development of bovine early embryos

“…The procedure for obtaining RNA probes was based on a protocol described previously 192 (Paradis et al 2005). Briefly, for the amplification of each selected candidate, a first round 193 of PCR was performed using specific primers (Table 2) on cDNA produced from bovine 194 oocytes as described above and the following conditions: denaturation at 95 °C for 10 min, 195 35 PCR cycles (denaturing: 94 °C for 1 min, annealing temperature (Table 1) …”

Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA

“…2A) by vortexing to avoid technical difficulties during microinjection of the dsRNA or water in the cytoplasm of the oocytes. Until used for microinjection, oocytes were held in tissue culture medium (TCM)-199 supplemented with 0.1% BSA (Sigma), 0.2 mM pyruvate and 50 mg/ml gentamycin sulphate (Sigma) in a humidified atmosphere with 5% CO 2 at 39 8C for 1-2 h. Prior to microinjection, immature oocytes were incubated for 20 min in TCM-199 supplemented with cytochalasin B at a final concentration of 8 mg/ml in order to reduce mechanical damage during injection (Paradis et al 2005). Subsequently, in three experimental replicates, a total of 935 immature oocytes were divided into three groups: C-mos dsRNA-injected (group 1, nZ327), water-injected group (group 2, nZ 303) and uninjected controls (group 3, nZ305).…”

“…Following its application in Caenorhabditis elegance (Fire et al 1998), the RNAi approach by introduction of dsRNA has been used in various mammalian species such as human (Brusselmans et al 2005, Cheng et al 2005, Hyslop et al 2005, mouse (Svoboda et al 2000, Wianny & ZernickaGoetz 2000, Grabarek et al 2002, Stein et al 2003, Alizadeh et al 2005, Gui & Joyce 2005, Plusa et al 2005) and porcine (Cabot & Prather 2003). Most recently, the work by our group (Nganvongpanit et al 2006) and others (Paradis et al 2005) have used the RNAi approach to suppress transcripts in bovine embryos and oocytes respectively. Recently, microinjection of dsRNA which interferes with genes that regulate cell polarity (Par3 and aPKC) in randomly selected blastomeres of cleavage-stage mouse embryos has enabled direction of defined cells to new positions and redirection of their fate in the preimplantation embryos (Plusa et al 2005).…”

Opportunities and challenges in applying genomics to the study of oogenesis and folliculogenesis in farm animals

“…At the moment, RNA interference represents the best hope for oocyte functional genomic studies. Since no promoters have been found to be active before the MET, it is not possible to use hairpin RNA; but since Dicer is present and active, and in absence of interferon response, both long and short double-stranded RNA can be used effectively in the bovine species (Paradis et al 2005) as in the mouse. Target genes should be carefully selected as a noticeable effect is expected only for genes, not for already present instable protein form.…”

“…Also, because of the long delay between oocyte injection and blastocyst formation, some late transcripts might be more difficult to knock down. Despite several publications on bovine oocytes (Paradis et al 2005, Nganvongpanit et al 2006, the resulting embryos are often developmentally compromised. Folliculogenesis and oogenesis seem integrated at least in the mouse, where knockouts have shown the multiple links between oocyte and follicle survival.…”