Mendez Flor M, Núñez Felipe J, Zorrilla-Veloz Rocío I, Lowenstein Pedro R, Castro Maria G
Department of Cell and Developmental Biology, University of Michigan Medical School.
Department of Cell and Developmental Biology, University of Michigan Medical School; Department of Neurosurgery, University of Michigan Medical School.
J Vis Exp. 2018 Jan 29(131):57016. doi: 10.3791/57016.
Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.
表观遗传修饰可能参与胶质瘤的发生和发展。癌基因和肿瘤抑制基因启动子及调控区域的甲基化和乙酰化变化可导致基因表达改变，并在脑肿瘤发病机制中起重要作用。天然染色质免疫沉淀（ChIP）是一种常用技术，可用于检测与DNA紧密结合的修饰或其他蛋白质。与交联ChIP不同，在天然ChIP中，细胞不用甲醛处理来使蛋白质与DNA共价连接。这具有优势，因为有时交联可能会固定那些仅与DNA瞬时相互作用且在基因调控中无功能意义的蛋白质。此外，抗体通常是针对未固定的肽产生的。因此，在天然ChIP中抗体特异性增加。然而，需要牢记的是，天然ChIP仅适用于研究与DNA紧密结合的组蛋白或其他蛋白质。本方案描述了对小鼠脑肿瘤神经球进行天然染色质免疫沉淀的方法。
