Establishing CRISPR interference-based genome-wide screening platform for identification of novel genes in macrophage alternative polarization

• Main Authors: Yi-Hsin Wu, 吳以新
• Other Authors: Hsin-Yue Tsai
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/m99x3g

碩士 === 國立臺灣大學 === 分子醫學研究所 === 107 === Macrophages are crucial players in immune regulation. They have a wide spectrum of activation states depend on the diverse surrounding stimuli they receive. Classical activation (M1) and alternative activation (M2) are described as two extremes of their polarized states, which elicit pro-inflammatory responses and anti-inflammatory responses respectively to maintain tissue homeostasis. Regnase-1 is a ribonuclease essential in controlling immune responses by regulating mRNA decay of proinflammatory cytokines, and it is reported to be important in promoting macrophage M2 polarization in which ER stress, ROS and autophagy are involved. However, detailed regulatory mechanism of this pathway is remained unclear. The goal of our study is to perform a genome-scale CRISPRi-dCas9 screening to explore new regulators in Regnase-1 mediated M2 polarization. By flow cytometry detection of M2 markers expression, we can identify genes that after CRISPRi disruption and Regnase-1 overexpression lead to decreased M2 expression, as potential regulators in this pathway. We have tested and compared the M2 phenotypes of four mice macrophage cell lines and examined the M1/M2 discrimination of several M2 markers by flow cytometry analysis. Our results demonstrated the M2 discriminating ability of Egr2 and CD206, which by flow cytometry detection can together be used to distinguish M2 phenotypes in both BMDMs and immortalized BMDMs. We have also established CRISPRi-Regnase-1 and inducible Regnase-1 overexpression system for further proof-of-principle screening and the preparations of the large-scale screening. Our data also infer a potential relation between ER stress related protein and M2 polarization, which is to be further investigated in the future works.