Establishment of RNA interference of a specific specific gene in bovine preimplantation embryos

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RNA interference (RNAi) is a strong tool to interfere a specific mRNA and widely applied for basic research for cell and tissue functions as well as medical treatment. Since the first report of RNAi in mouse preimplantation embryos (1999), RNAi research of gene regulation in mammalian embryos has started. However, RNAi in livestock animals such as bows and pigs has not been fully investigated. Therefore, in the present project, we investigated the efficiency of RNA interference on early development and tissue cell functions of bovine preimplantation embryos and cells. In the first experiment, we tried to establish the RNAi of a bovine specific gene in preimplation embryos and somatic cells. We used a dicer gene that has an important role for the initiation of RNAi to cut the double-stranded RNA to form the RNA-induced silencing complex (RISC). Knockout of dicer gene caused the lethal effect for the differentiation of mouse germ cells. We synthesized short interfering RNA (siRNA) that m … More atches the axling region for helicase domain in dicer gene. Interfering effect of dicer siRNA was investigated by introducing to cultured bovine cumulus and oviductal epithelial cells. After 24h, mRNA levels were significantly decreased in both cells. After confirming the interfering effect of synthesized dicer siRNA in tissue cells, siRNA was microinjected to bovine 1-cell stage embryos. For introduction of dimsr siRNA, less than 5 pl of 250 uM of siRNA solution was microinjected. After 24 of introduction, mRNA was significantly reduced; however, rate of division was not affected. Immunostaining of dicer also revealed the decrease in the dicer protein expression by siRNA. In the additional research, we invested the RNAi in cycrooxygenase-2 (Cox-2) gene in cumulus cells. Introduction of Cox-2 siRNA significantly decreased the mRNA levels 12h after transfection. Prostaglandin F2 α (PGF2α) that is synthesized by cox-2, in culture medium was also decreased 24h after siRNA introduction.In the second experiment, we investigated the establishment of interference in more developed stage of bovine embryos. RNAi research in mammalian embryos has been achieved using only oocytes and 1-cell stage embryos because of the difficulty in the introduction of siRNA or dsRNA solution into divided blastomeres. Therefore we tried to introduce the siRNA solution into more developed stage of embryos using lipofection method. FTTC-labeled negative siRNA was introduced to 1, 8, morula and blastocyst stage embryos. After transfection, fluorescence was detected only in zona pellucidae in all stage embryos by binding of siRNA-lipofection conjugate with zona pellucidae and no introduction of siRNA into embryos was observed. Therefore, zona free embryos were used for siRNA introduction. High fluorescence intensity was detected in zona-free embryos at all stages used for the experiment After introduction of FITC-siRNA with lipofection, no toxicity on embryo development was observed except for the lower development up to 8-16 cell stages by the insufficient division and 3-dimentional localization of blastmeres without zona pellucia. Dicer mRNA was significantly decreased 8-cell stage embryos after 24th of introduction with dicer siRNA by lipofection. These results indicated the RNAi of specific gene can be carried out using developed-stage embryos. Less