奶牛单胚RT—PCR方法的建立.pdfVIP

2007年 lO月 甘 肃 农 业 大 学 学 报 第 4 2卷 第5期37～4O JOURNAL OF GANSU AGRICULTURAL UNIVERSITY 双 月 刊 奶牛单胚RT—PCR方法的建立 张进隆 ，赵兴绪 ，陶金 忠 ， 杨永新 ，张 勇 (1．甘肃农业大学动物医学院。甘肃兰州 730070；2．甘肃畜牧工程职业技术学院，甘肃 武威 733006) 摘要：单个胚胎RT—PCR法在附植前胚胎发育基因的研究中具有重要作用。选取体外培养的4细胞期胚 胎。用酸性台氏液除去透明带。胚胎裂解液裂解胚胎。以单胚和多胚mRNA作模板。用锚定引物和随机引物进行 RT～PCR扩增．用特异引物进行持家基因GAPDH扩增作对照，分别用琼脂糖凝胶电泳和聚丙烯酰胺凝胶电泳比 较单胚与多胚扩增结果。结果表明。单胚能够达到与多胚同样的结果，从单胚扩增物检出GAPDH。因此建立的单胚 RT～PCR方法是有效的． 关键词：奶牛；单胚；RT—PCR；建立 中图分类号：S 823．9 文献标识码：A 文章编号：1003-4315(2007)06-0037—04 Development of the single bovine embryo reverse transcription polymerase chain reaction ZHANG Jin—long ’。，ZHAO Xing—XU ，TAO Jin—zhong ，YANG Yong—xin ，ZHANG Yong (1．College of Veterinary Medicine，Gansu Agricultural University，Lanzhou 730070。China 2．Gansu Vocational College of Animal Husbandry and Egineering．Wuwei 733006，China) Abstract：The single embryo RT—PCR play an important role in investigating preimplantation devel— opment—related genes．In the current research，4 cell embryos cultured in vitro were selected，zone pellucida was lysised with acidic Tyrodes solution，embryos were lysised with lysis buffer，single embryo and multi— pie embryos mRNA were used as template，PCR amplification was conducted with radom primer and oligo 一 (dT)and the house keeping gene GAPDH PCR amplification as control，amplification products were se— prated on a agrose gel electrophoresis(AGE)and polyacrylamide gel electrophoresis(PAGE)，multiple and single embryo had the same expression results，the GAPDH was detected successfully in single embryo． Thus，the method of single embryo RT—PCR was effective． Key words：COW；single embryo；RT—PCR；establishment 哺乳动物早期胚胎的发育需要经过数次卵裂、 发育阶段，胚胎基因表达模式和特性不同，应用各种 胚胎基因组的活化、囊胚的形成等重要阶段，在不同 试验方法