Voss Anne K, Dixon Mathew P, McLennan Tamara, Kueh Andrew J, Thomas Tim
The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia.
Methods Mol Biol. 2012;809:335-52. doi: 10.1007/978-1-61779-376-9_23.
During prenatal development, a large number of different cell types are formed, the vast majority of which contain identical genetic material. The basis of the great variety in cell phenotype and function is the differential expression of the approximately 25,000 genes in the mammalian genome. Transcriptional activity is regulated at many levels by proteins, including members of the basal transcriptional apparatus, DNA-binding transcription factors, and chromatin-binding proteins. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency, with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method to assess if chromatin modifications or proteins are present at a specific locus. ChIP involves the cross linking of DNA and associated proteins and immunoprecipitation using specific antibodies to DNA-associated proteins followed by examination of the co-precipitated DNA sequences or proteins. In the last few years, ChIP has become an essential technique for scientists studying transcriptional regulation and chromatin structure. Using ChIP on mouse embryos, we can document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development. Here, we describe a ChIP technique adapted for mouse embryos.
在产前发育过程中,会形成大量不同的细胞类型,其中绝大多数细胞含有相同的遗传物质。细胞表型和功能的巨大多样性的基础是哺乳动物基因组中约25000个基因的差异表达。转录活性在多个水平上受到蛋白质的调控,这些蛋白质包括基础转录装置的成员、DNA结合转录因子和染色质结合蛋白。重要的是,染色质结构决定了特定基因组位点用于转录激活的可用性以及转录发生的效率。染色质免疫沉淀(ChIP)是一种评估特定位点是否存在染色质修饰或蛋白质的方法。ChIP包括DNA与相关蛋白质的交联以及使用针对DNA相关蛋白质的特异性抗体进行免疫沉淀,随后检测共沉淀的DNA序列或蛋白质。在过去几年中,ChIP已成为研究转录调控和染色质结构的科学家的一项重要技术。通过对小鼠胚胎进行ChIP,我们可以记录哺乳动物发育过程中体内基因组位点上特定蛋白质和染色质修饰的存在与否。在这里,我们描述一种适用于小鼠胚胎的ChIP技术。