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

RNA干扰(RNAi)是干扰特定mRNA的有力工具，被广泛应用于细胞和组织功能的基础研究以及医疗领域。自从1999年首次报道了小鼠着床前胚胎中的RNAi以来，关于哺乳动物胚胎中基因调控的RNAi研究已经开始。然而，在弓和猪等家畜中的RNAi还没有得到充分的调查。因此，在本项目中，我们研究了RNA干扰对牛着床前胚胎和细胞早期发育和组织细胞功能的影响。在第一个实验中，我们试图在植入前的胚胎和体细胞中建立一个牛特有基因的RNAi。我们使用在RNAi启动中起重要作用的DICER基因来切割双链RNA，形成RNA诱导沉默复合体(RISC)。DICER基因敲除对小鼠生殖细胞的分化有致死作用。我们合成了m…的短干扰rna更接近于DICER基因中解旋酶结构域的轴区。将小分子siRNA导入体外培养的牛卵丘和输卵管上皮细胞，研究其对卵丘细胞的干扰作用。24小时后，两种细胞的mRNA水平均显著下降。在证实合成的小分子siRNA对组织细胞的干扰作用后，将siRNA显微注射到牛1细胞期胚胎中。对于引入dimsr siRNA，显微注射少于5pl的250um siRNA溶液。导入24小时后，mRNA量显著减少，但不影响分裂率。免疫组织化学显示siRNA可降低DICER蛋白的表达。在另一项研究中，我们将RNAi导入卵丘细胞中的环氧合酶-2(COX-2)基因。COX-2 siRNA的引入显著降低了转染后12h的mRNA水平。培养上清液中由COX-2合成的前列腺素F_2α(PGF_2α)在小干扰RNA导入后24小时也显著降低。由于在分裂的卵裂球中引入siRNA或dsRNA溶液的困难，哺乳动物胚胎中的RNAi研究仅使用卵母细胞和1-细胞期胚胎。因此，我们尝试用脂质体转染法将siRNA溶液引入胚胎发育的更高阶段。将FTTC标记的阴性siRNA分别导入1、8、桑拿期和囊胚期胚胎。转染后，siRNA-脂质体结合物与透明带结合，仅在各发育阶段胚胎的透明带中检测到荧光，未观察到siRNA导入胚胎。因此，无透明带胚胎被用于siRNA的导入。将FITC-siRNA导入脂质体后，在去透明带胚胎的各个发育阶段均检测到较高的荧光强度，对胚胎的发育没有毒性，但由于无透明带的胚泡分裂和三维定位不充分，导致胚胎发育降低到8-16个细胞期。Dicer siRNA通过脂质体导入8-细胞期胚胎24小时后，8-细胞期胚胎的DICER基因表达水平显著降低。这些结果表明，可以利用发育阶段的胚胎进行特定基因的RNAi。较少

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