Cell type-specific expression profiting in plants via cell sorting of protoplasts from fluorescent reporter lines
Cell type-specific expression profiting in plants via cell sorting of protoplasts from fluorescent reporter lines
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DOI：
10.1038/nmeth0805-615
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发表时间：
2005-08-01
期刊：
影响因子：
48
通讯作者：
Benfey, PN
中科院分区：
文献类型：
--
作者：
Birnbaum, K;Jung, JW;Benfey, PN
为了研究发育事件和基因表达之间的关系，细胞特异性的基因活性解析是至关重要的。在基因组水平上很难获得如此高分辨率的数据，因为首先需要分离细胞，然后必须收集或随后扩增足够数量的信使核糖核酸，以便进行大规模的盈利分析。基因组学方法在推断发育回路方面具有巨大潜力，并与遗传工具相结合，可以发现已知发育调控因子的未知下游靶点。我们已经开发出一种方法，可以用来分离多达数十万个特定细胞类型的植物细胞，纯度非常高，然后可以用于微阵列分析。该方法利用在组织学定义的细胞类型中表达绿色荧光蛋白(GFP)的报告基因，目前已有大量收集(表1)。GFP感兴趣线被放大，组织被收集并快速转化为原生质体1(1，2)。然后使用荧光激活细胞分类器(FACS)分离GFP阳性细胞。总RNA被分离，使用标准程序标记，并应用于微阵列(图1)。这项技术已经被用来生成拟南芥根中细胞类型和组织的表达谱(3)，尽管它可以用于任何细胞瓦特容易被消化的组织。这里提供的协议已经针对根进行了优化。
To investigate the relationship between developmental events and gene expression, cell-specific resolution of gene activity is critical. Such high-resolution data have been difficult to obtain at a genomic level because cells first need to be isolated, and then sufficient amounts of mRNA must be collected, or subsequently amplified, for a Large-scale profiting analysis. Genomics methods have tremendous potential to infer developmental circuits and, in combination with genetic tools, to discover the unknown downstream targets of known developmental regulators. We have developed a method that can be used to isolate up to hundreds of thousands of plant cells of a specific cell type, with very high purity, which can then be used for microarray analysis. The method makes use of reporter Lines expressing green fluorescent protein (GFP) in histologically defined cell types, of which Large collections are now available (Table 1). The GFP Line of interest is bulked and the tissue is collected and rapidly converted into protoplastsl(1,2). GFP-positive cells are then isolated using a fluorescence-activated cell sorter (FACS). Total RNA is isolated, Labeled using standard procedures and applied to microarrays (Fig. 1). The technique has been used to generate expression profiles of cell types and tissues in the Arabidopsis thaliana root(3), although it can be used for any tissue whose cell watts can be readily digested. The protocol presented here has been optimized for roots.