Bargmann Bastiaan O R, Birnbaum Kenneth D
Department of Biology, New York University, Center for Genomics and Systems Biology, USA.
J Vis Exp. 2010 Feb 18(36):1673. doi: 10.3791/1673.
High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.
对基因表达进行高分辨率、细胞类型特异性分析，能极大地增进我们对任何多细胞生物发育调控及对环境刺激反应的理解。原位杂交和报告基因可视化在一定程度上可用于此目的，但对于高分辨率定量逆转录聚合酶链反应（RT-PCR）或全转录组高通量分析而言，从特定细胞类型中分离RNA是必不可少的。在特定细胞类型中表达荧光蛋白标记的组织进行细胞解离，随后进行荧光激活细胞分选（FACS），就能够收集到足够量的材料用于RNA提取、互补DNA（cDNA）合成/扩增以及微阵列分析。植物研究群体可获取大量细胞类型特异性荧光报告株系。在此，使用了拟南芥根的两个标记株系：P(SCR;)::GFP（内皮层和静止中心）和P(WOX5;)::GFP（静止中心）。大量（数千株）幼苗通过水培或在琼脂平板上生长，然后收获以获得足够的根材料用于进一步分析。植物材料的细胞解离是通过对细胞壁进行酶解来实现的。该过程利用高渗透压诱导的质壁分离以及市售的纤维素酶、果胶酶和半纤维素酶，将原生质体释放到溶液中。对绿色荧光蛋白（GFP）阳性细胞进行FACS时，利用488纳米激光激发原生质体后其绿色与红色发射光谱的可视化。GFP阳性原生质体可通过其绿色与红色发射比例的增加来区分。原生质体通常直接分选到RNA提取缓冲液中，并储存起来以便后续进一步处理。该技术被证明是直接且可行的。此外，研究表明，即使对于非常稀少的细胞类型（如静止中心细胞），它也能毫无困难地用于分离足够数量的细胞进行转录组分析。最后，展示了一种拟南芥幼苗的生长设置，该设置能够在细胞分选之前对植物进行简单处理（例如用于生物或非生物胁迫反应的细胞类型特异性分析）。还讨论了植物原生质体FACS的潜在补充用途。
