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强力霉素诱导型Cas9基因编辑小鼠胚胎干细胞系的构建
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作者 姚玉琛 朱少华 +3 位作者 曹佳妮 张琳 杨月伟 赵同标 《发育医学电子杂志》 2018年第3期160-165,共6页
目的建立强力霉素(doxmycin,Dox)诱导型Cas9基因编辑小鼠胚胎干细胞系,以满足不同条件下基因编辑的实验需求。方法根据四环素(tetracycline,Tet)-on诱导表达调控系统的原理,分别构建含有调控元件反义Tet转录激活因子(reverse tetracy-cl... 目的建立强力霉素(doxmycin,Dox)诱导型Cas9基因编辑小鼠胚胎干细胞系,以满足不同条件下基因编辑的实验需求。方法根据四环素(tetracycline,Tet)-on诱导表达调控系统的原理,分别构建含有调控元件反义Tet转录激活因子(reverse tetracy-cline transcriptional activator,rt TA)的表达质粒p CDH-CAG-rt TA-IRES-m Cherry和含有Tet应答元件(Tet-responsive element,TRE)及Cas9的表达质粒p Tight-Cas9-IRES-GFP-Tight-Puro,其中红色荧光蛋白m Cherry通过内部核糖体进入位点(internal ribosome entry site,IRES)与rt TA表达,绿色荧光蛋白(green fluorescent protein,GFP)通过IRES与Cas9相连,分别指示rt TA与Cas9的表达,Puro抗性基因作为药物筛选标记。将上述两种质粒包装为慢病毒,分步转入小鼠胚胎干细胞中,建立Dox诱导Cas9表达的小鼠胚胎干细胞系。结果首先通过瞬时转染方法在293T细胞中验证其可诱导性,然后结合报告荧光与药物筛选获得同时整合有调控元件rt TA与应答元件TRE及Cas9的小鼠胚胎干细胞系,该细胞系加入Dox后出现有GFP报告荧光表达,实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,rt-q PCR)结果同时表明Cas9可以被成功诱导表达。结论本研究成功构建了Dox诱导Cas9表达的小鼠胚胎干细胞系,该细胞系在基因编辑方法上更加灵活可控,具有很高的潜在应用价值。 展开更多
关键词 CRISPR/Cas9 四环素诱导 条件性敲除 小鼠es细胞系
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Establishment of Embryonic Stem Cell Lines Derived from Outbred Mouse Embryos and Production of Chimeras 被引量:2
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作者 何维 高建刚 +1 位作者 刘晓 孙方臻 《Developmental and Reproductive Biology》 1996年第2期21-25,共5页
The aim of the present investigation was to determine if embryonic stem (ES) cells could be isolated from outbred mouse embryos (KM) and if chimeras could be producedly using outbred ES cells. Three ES cell lines,desi... The aim of the present investigation was to determine if embryonic stem (ES) cells could be isolated from outbred mouse embryos (KM) and if chimeras could be producedly using outbred ES cells. Three ES cell lines,designated KE1, KE2, and KE5,were isolated from 5 Kunming albino blastocysts. Normal diploid composition of these cell lines was above 70%. By using C57BL/6J and 615 blastocysts as host embryos, one chimera was obtained in living pups. It was shown for the first time that chimeras can be produced by using outhred ES cells. This work implies that to establish ES cell lines from other animal embryos is possible. More interestingly, white color dots from ES cells on the coat of this chimeric mouse enlarged a lot after half a year, indicated that ES cells were inhibited by cells from outbred mouse or the cells of outbred mouse grew vigiously. 展开更多
关键词 KM mouse blastocyst es cell line chimeric mice.
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Establishment of Embryonic Stem Cell Lines from C57BL/6J Mice and Generation of Chimeras
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作者 何维 高建刚 +1 位作者 刘晓 孙方臻 《Developmental and Reproductive Biology》 1997年第2期13-20,共8页
Four embryonic stem (ES) cell lines, designated CE1, CE2, CE3 and CE4, were isolated from C57BL/6J blastocysts. The ratio of normal diploid composition of these cell lines is above 70%. To examine the differentiation... Four embryonic stem (ES) cell lines, designated CE1, CE2, CE3 and CE4, were isolated from C57BL/6J blastocysts. The ratio of normal diploid composition of these cell lines is above 70%. To examine the differentiation potential of the ES cells, the CE2 cells were injected subcutaneously into syngenic mice, and many kinds of differentiated cells were observed on the sections of the teratoma derived from this ES cell line. On the other hand, to test the chimeric ability of the ES cells, the CE2 cells were microinjected into the blastocysts of ICR mice, and a chimera was obtained among living pups. These results show that CE2 ES cells are pluripotent stem cells, which can differentiate into many kinds of cell types, and can be used as a cell system for further research. 展开更多
关键词 C57BL/6J mouse es cell line establishment chimera.
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