Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice
Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice
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DOI：
10.1073/pnas.1734197100
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发表时间：
2003-09-30
影响因子：
11.1
通讯作者：
Smith, A
中科院分区：
文献类型：
--
作者：
Aubert, J;Stavridis, MP;Smith, A
转录因子Sox 1是哺乳动物神经祖细胞最早和最特异的已知标记。在胎儿发育过程中，Sox 1在整个中枢神经系统中由增殖的祖细胞表达，除了透镜外，没有组织表达。我们产生了一个报告小鼠系，其中egfp被插入到Sox 1基因座。Sox 1（GFP）动物忠实地再现了内源基因的表达。我们已经使用GFP报告纯化神经上皮细胞从胚胎10.5天的胚胎荧光激活细胞分选。然后使用从Sox 1（GFP+）和Sox 1（GFP-）胚胎细胞制备的RNA对从分化的胚胎干细胞制备并排列在玻璃芯片上的消减cDNA进行初步筛选。15个独特的差异表达的基因被确定，所有以前与胎儿或成人神经组织。针对先前未知的胚胎表达的两个基因Lrrn 1和Musashi 2的全胚原位杂交证实了该筛选对早期神经外胚层标记物的选择性。
The transcription factor Sox1 is the earliest and most specific known marker for mammalian neural progenitors. During fetal development, Sox1 is expressed by proliferating progenitor cells throughout the central nervous system and in no tissue but the lens. We generated a reporter mouse line in which egfp is inserted into the Sox1 locus. Sox1(GFP) animals faithfully recapitulate the expression of the endogenous gene. We have used the GFP reporter to purify neuroepithelial cells by fluorescence-activated cell sorting from embryonic day 10.5 embryos. RNAs prepared from Sox1(GFP+) and Sox1(GFP-) embryo cells were then used to perform a pilot screen of subtracted cDNAs prepared from differentiating embryonic stem cells and arrayed on a glass chip. Fifteen unique differentially expressed genes were identified, all previously associated with fetal or adult neural tissue. Whole mount in situ hybridization against two genes of previously unknown embryonic expression, Lrrn1 and Musashi2, confirmed the selectivity of this screen for early neuroectodermal markers.