来自霍德华休斯医学院,西雅图弗莱德哈金森癌症研究中心的研究人员提出了一种可以用于分离特异组织细胞的简单方法,这有利于更加方便的进行不同组织类型的基因表达和染色体修饰分析。这一研究成果公布在Cell出版社旗下的Developmental Cell杂志上,并获得了F1000多位专家的联合推荐。
领导这一研究的是弗莱德哈金森癌症研究中心的Steven Henikoff教授,这位科学家在研究技术方面获得了多项突破,曾发明了TILLING技术,这一全新的反向遗传学技术已经应用于多种生物中,如拟南芥、玉米、水稻、百脉根、小鼠、斑马鱼、果蝇、线虫等,可以通过高通量检测方法,快速有效的从化学诱变剂EMS诱变过的突变群体中鉴定处点突变,TILLING技术开创了一种高通量,低成本反向遗传的新局面,对于未来的医学检测,生物制药等方面具有重要的意义。
而在这篇题为“A Simple Method for Gene Expression and Chromatin Profiling of Individual Cell Types within a Tissue”的最新文章中,Henikoff教授提出了另外一种新方法,可以分离特异组织细胞的简单方法。
研究分析发育过程中的特殊细胞的功能和生长情况,需要分离不同细胞类型的细胞,目前常用的组织细胞分检方法需要许多复杂的设备,比如显微镜和流式细胞仪等,而且过程繁琐,对于研究人员来说是一件费时费力的工作。
Henikoff教授发现了一种能在体内标记并分离特殊细胞类型细胞核的简单方法,这种方法利用一种融合了生物素配基识别多肽的核被膜靶向序列,通过在目标细胞中表达,从而达到识别的目的,这能让研究人员更加方便的进行不同组织类型的基因表达和染色体修饰分析。
据称利用这种方法分拣细胞只需要用磁珠,比如说选定一种可以转化的模式生物,然后设计一个核膜蛋白的质粒(由组织特异性启动子、显色蛋白(如GFP)、亲和性结构(如biotin)、核膜蛋白组成),质粒转化后分离细胞,提取组织总细胞核,然后用识别biotin的磁珠吸引分离那些特异表达核膜蛋白的细胞核,这样就能进行不同类型的细胞的基因表达,染色体方面的研究了。
这篇文章就是以拟南芥为例,利用这种方法进行了分离,从中发现了上百个特殊类型细胞中倾向表达的基因,继而了解了毛细胞与非毛细胞的基因表达差异。
原文出处:
Developmental Cell doi:10.1016/j.devcel.2010.05.013
A Simple Method for Gene Expression and Chromatin Profiling of Individual Cell Types within a Tissue
Highlights
An affinity-based method was developed to purify nuclei from specific cell types
High yield and purity of nuclei were obtained from each of the tested cell types
Gene expression and chromatin profiles of individual root cell types were generated
This method does not require specialized equipment and should be widely applicable
Summary
Understanding the production and function of specialized cells during development requires the isolation of individual cell types for analysis, but this is currently a major technical challenge. Here we describe a method for cell type-specific RNA and chromatin profiling that circumvents many of the limitations of current methods for cell isolation. We used in vivo biotin labeling of a nuclear envelope protein in individual cell types followed by affinity isolation of labeled nuclei to measure gene expression and chromatin features of the hair and non-hair cell types of the Arabidopsis root epidermis. We identified hundreds of genes that are preferentially expressed in each cell type and show that genes with the largest expression differences between hair and non-hair cells also show differences between cell types in the trimethylation of histone H3 at lysines 4 and 27. This method should be applicable to any organism that is amenable to transformation.