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作 者:戴星[1] 杨康平[1] 尹战海[1] 马巍[1] 杨益民[1] 周晓玲[1] 姚璐 DAI Xing;YANG Kangping;YIN Zhanhai;MA Wei;YANG Yimin;ZHOU Xiaoling;YAO Lu(The First Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710061,China)
机构地区:[1]西安交通大学第一附属医院,西安710061 [2]西安交通大学医学部基础医学院
出 处:《山东医药》2019年第33期1-4,共4页Shandong Medical Journal
基 金:国家自然科学基金资助项目(81702669;31300899);陕西省重点研发计划资助项目(2017SF-231);陕西省自然科学基金资助项目(2018JM7035);中央高校基本科研业务费项目(XJJ2018046);2019年陕西省大学生创新创业训练计划项目;2018年西安交通大学医学部本科开放实验项目
摘 要:目的以人骨肉瘤细胞的肾上腺髓质素(ADM)为作用靶点,建立以慢病毒为载体的RNA干扰体系,以高效抑制ADM基因的表达。方法构建慢病毒载体,设计3条针对ADM基因序列的shRNA序列(shRNA-1、2、3),靶向ADM shRNA与慢病毒载体连接后,挑选LV-ADM-shRNA阳性克隆进行PCR鉴定并测序。经测序确认寡核苷酸插入部位正确后,提取目的质粒LV-ADM-shRNA 1、2、3,在293T细胞中包装携带目的质粒的慢病毒,然后采用多孔稀释法测定慢病毒滴度。结果构建3条靶向ADM的LV-shRNA慢病毒载体,并获得较高滴度的慢病毒颗粒。结论成功建立以慢病毒为载体的人骨肉瘤细胞ADM RNA干扰体系。Objective To establish the lentivirus RNA interference system targeting adrenomedullin(ADM)of human osteosarcoma cells to efficiently inhibit the expression of ADM gene.Methods We constructed lentivirus vector and designed three shRNA sequences(shRNA-1,2,3)for ADM gene sequence.When the target ADM shRNA was connected to the lentivirus vector,LV-ADM-shRNA positive clones were selected for PCR identification and sequencing.After the oligonucleotide insertion sites were confirmed to be correct by sequencing,the target plasmids(LV-ADM-shRNA 1,2,3)were extracted.The lentiviruses were packaged in 293T cells,and the titer of lentivirus was determined by porous dilution method.Result Three lentiviral vectors targeting ADM-shRNA were constructed and lentiviral particles with higher titers were obtained.Conclusion We successful established the RNA interference system with lentivirus-mediated adrenomedullin in human osteosarcoma cells.
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