Production of green fluorescent protein transgenic embryonic stem cells using the GENSAT bacterial artificial chromosome library
Production of green fluorescent protein transgenic embryonic stem cells using the GENSAT bacterial artificial chromosome library
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DOI：
10.1634/stemcells.2006-0173
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发表时间：
2007-01-01
期刊：
影响因子：
5.2
通讯作者：
Studer, Lorenz
中科院分区：
文献类型：
--
作者：
Tomishima, Mark J.;Hadjantonakis, Anna-Katerina;Studer, Lorenz
转基因绿色荧光蛋白（GFP）报告胚胎干细胞（ES细胞）是研究发育过程中基因调控和谱系选择的有力工具。在此我们介绍一种在选定基因控制下产生表达GFP的ES细胞的快速方法。对来自先前构建的GFP转录融合文库（基因表达神经系统图谱[GENSAT]）的细菌人工染色体（BAC）进行改造以用于ES细胞，并产生了多种BAC转基因ES细胞系。在神经分化过程中，通过Hes5、Dll1和GFAP分别标记神经干细胞、神经元前体细胞和神经胶质后代，证实了转基因细胞系中特异性的GFP表达。GFP在ES细胞后代中受到动态调控，以响应抑制Notch信号的可溶性因子以及一种决定星形胶质细胞命运选择的因子。我们的实验方案提供了一种简单有效的策略，利用整个GENSAT BAC文库创建数百种新型荧光细胞系，用于ES细胞生物学研究。
Transgenic green fluorescent protein (GFP) reporter embryonic stem (ES) cells are powerful tools for studying gene regulation and lineage choice during development. Here we present a rapid method for the generation of ES cells expressing GFP under the control of selected genes. Bacterial artificial chromosomes (BACs) from a previously constructed GFP transcriptional fusion library (Gene Expression Nervous System Atlas [GENSAT]) were modified for use in ES cells, and multiple BAC transgenic ES cell lines were generated. Specific GFP expression in transgenic cell lines was confirmed during neural differentiation marking neural stem cells, neuronal precursors, and glial progeny by Hes5, Dll1, and GFAP, respectively. GFP was dynamically regulated in ES cell progeny in response to soluble factors that inhibit Notch signaling and a factor that directs astroglial fate choice. Our protocols provide a simple and efficient strategy to utilize the whole GENSAT BAC library to create hundreds of novel fluorescent cell lines for use in ES cell biology.