背景:Survivin在多种肿瘤组织中的高表达,具有调节细胞增殖分裂和强大的抗凋亡功能。目的:利用RNA干扰技术,构建具有特异性阻断C57BL小鼠survivin基因的微小RNA(micro RNA,miRNA)表达载体。设计、时间及地点:单一样本观察,于2008-06/11...背景:Survivin在多种肿瘤组织中的高表达,具有调节细胞增殖分裂和强大的抗凋亡功能。目的:利用RNA干扰技术,构建具有特异性阻断C57BL小鼠survivin基因的微小RNA(micro RNA,miRNA)表达载体。设计、时间及地点:单一样本观察,于2008-06/11在重庆医科大学附属第一医院神经内科实验中心完成。材料:环形pcDNATM6.2-GW/EmGFPmiR和BLOCK-iTTM PolII miR RNA干扰Expression Vector Kit with EmGFP为Invitrogen公司产品;DH5a大肠杆菌为实验室保存;xhoI和BamHI酶、壮观霉素均为上海生工生物工程有限公司产品。方法:应用设计软件在C57BL小鼠survivin基因的mRNA上寻找特异性的短核苷酸序列,并设计合成4对寡核苷酸序列,经退火后形成双链DNA片段,采用基因克隆技术,将其克隆到pcDNATM6.2-GW/EmGFPmiR的载体中,转化DH5a大肠杆菌,挑单菌落种于含有壮观霉素LB液体培养基中,提取质粒。主要观察指标:应用测序法和琼脂糖电泳检测对重组体进行鉴定。结果:测序结果显示插入片段与线性载体连接正确,无碱基突变、缺失、插入等异常。双酶切处理pcDNATM6.2-GW/EmGFP-miR重组质粒结果显示片段大小与预期相符。结论:实验结果表明成功构建了C57BL小鼠survivin基因的微小RNA表达载体。展开更多
A new method for establishing ES cell lines from 129/ter、C57BL/6J mice was set up which was characterized by the murine embryonic fibroblast cell(MEF) feeder, the medium of rat heart cell-conditioned medium(RH-CM)for...A new method for establishing ES cell lines from 129/ter、C57BL/6J mice was set up which was characterized by the murine embryonic fibroblast cell(MEF) feeder, the medium of rat heart cell-conditioned medium(RH-CM)for ES cells, and the consecutive digestion by the digestion liquid containing 1% serum. Every group of improved experiments was done with a control of routine method. The results showed that, compared with routine method, the improved way increased the ratio of ES cell lines of 129/ter mice from 11.8% to 33.3%, and of C57BL/6J from 3.7% to 13.3%. The difference is distinct. The passage culture of ES cells showed that, compared with medium added LIF, RH-CM not only inhibited the differentiation of murine ES cells, maintained its dipoild karyotype, but also promote its adherence growth. This kind of culture condition not only maintained the ES cells in an undifferentiated state and their normal dipoild karyotype, but also a series of other characteristics of totipotent embryonic stem cells during extended culture period.展开更多
文摘背景:Survivin在多种肿瘤组织中的高表达,具有调节细胞增殖分裂和强大的抗凋亡功能。目的:利用RNA干扰技术,构建具有特异性阻断C57BL小鼠survivin基因的微小RNA(micro RNA,miRNA)表达载体。设计、时间及地点:单一样本观察,于2008-06/11在重庆医科大学附属第一医院神经内科实验中心完成。材料:环形pcDNATM6.2-GW/EmGFPmiR和BLOCK-iTTM PolII miR RNA干扰Expression Vector Kit with EmGFP为Invitrogen公司产品;DH5a大肠杆菌为实验室保存;xhoI和BamHI酶、壮观霉素均为上海生工生物工程有限公司产品。方法:应用设计软件在C57BL小鼠survivin基因的mRNA上寻找特异性的短核苷酸序列,并设计合成4对寡核苷酸序列,经退火后形成双链DNA片段,采用基因克隆技术,将其克隆到pcDNATM6.2-GW/EmGFPmiR的载体中,转化DH5a大肠杆菌,挑单菌落种于含有壮观霉素LB液体培养基中,提取质粒。主要观察指标:应用测序法和琼脂糖电泳检测对重组体进行鉴定。结果:测序结果显示插入片段与线性载体连接正确,无碱基突变、缺失、插入等异常。双酶切处理pcDNATM6.2-GW/EmGFP-miR重组质粒结果显示片段大小与预期相符。结论:实验结果表明成功构建了C57BL小鼠survivin基因的微小RNA表达载体。
文摘A new method for establishing ES cell lines from 129/ter、C57BL/6J mice was set up which was characterized by the murine embryonic fibroblast cell(MEF) feeder, the medium of rat heart cell-conditioned medium(RH-CM)for ES cells, and the consecutive digestion by the digestion liquid containing 1% serum. Every group of improved experiments was done with a control of routine method. The results showed that, compared with routine method, the improved way increased the ratio of ES cell lines of 129/ter mice from 11.8% to 33.3%, and of C57BL/6J from 3.7% to 13.3%. The difference is distinct. The passage culture of ES cells showed that, compared with medium added LIF, RH-CM not only inhibited the differentiation of murine ES cells, maintained its dipoild karyotype, but also promote its adherence growth. This kind of culture condition not only maintained the ES cells in an undifferentiated state and their normal dipoild karyotype, but also a series of other characteristics of totipotent embryonic stem cells during extended culture period.
