| Macrophages play an essential role in our body’s immune system. Their primary functions include phagocytosis and elimination of harmful bacteria, parasite, cancerous cells as well as cells that undergo apoptosis and senescence. Macrophages could also maintain the defence mechanisms of our immune system. Lymphocyte recruitment and antigen presentation. Macrophages are widely distributed throughout the body. Before they differentiate into tissue-specific mature macrophages, pro-macrophages originate from monocytes that could migrate through the inner wall of blood vessels to different tissues such as liver, spleen, lung, CNS and bone. Take the alveolar macrophages for example. When pathogenic substances invade, the pattern recognition receptors(PRR) on the surfaces of the alveolar macrophages, i.e. the toll-like-receptors, will combine with corresponding ligands so as to improve the phagocytic ability of the macrophages. PRR plays a very important role in giving play to the phagocytic function of the macrophages in a way of recognizing the specificity area of the pathogenic substances and then phagocytising the pathogenic substances. PRR on the surfaces of the macrophages are mainly divided into three types: the main function of the first type is to serve as the activating complement, like mac1; the second type of receptors can combine and phagocytise antigens, like the toll-like receptor; and the third type of receptors are featured by the signal molecules and can influence genetic transcription activity after being activated.The phagocytic function of the macrophages plays a pivotal role in exerting its immune defence effect. According to current researches, the phagocytic function of the macrophages is closely related to the occurrence of many diseases. Take the neurodegenerative disease as example. Currently, it is believed that the microglial cells, which can remove substances like impaired nerves, apoptotic cells and plaques, are the key to the pathogenesis of the neurodegenerative disease. Microglial cells can remove substances like impaired nerves, apoptotic cells and plaques in the central nervous system, and diseases will be caused if the removal functions are abnormal. However, the regulation mechanism of the phagocytic function of the macrophages is unclear at present. Constantly focusing on the study on macrophages, our laboratory plans to screen the key genes which regulate the phagocytic function of macrophages at the whole genome level, so as to provide clues and thoughts for the subsequent mechanism study. The introduction of the CRISPR technology in 2013 brings great convenience to our study on screening work. CRISPR/Cas9 technology, the revolutionary novel genome editing technology emerging in recent years, can realize efficient gene editing at the cellular level. Due to the multifunctional and programmable characteristics, the CRISPR/Cas9 technology can build the rat disease models more quickly. At the same time, the CRISPR/Cas9 technology shows a huge application potential in the research, treatment and prevention of many diseases such as the genetic diseases and malignant tumors.The advancement of CRISPR/Cas9 technology has proved its applications in scientific research and medicine. To facilitate research collaboration and better quality research, researchers at the core frontier of this technology have kindly provided CRISPR/Cas9 libraries to Addgene Inc. for global demand. Therefore, we aim to discover and identify genes that are important for phagocytosis by using a CRISPR/Cas9 library screen. There are two prerequisites for this screen: 1) a library that covers enough genome information and 2)optimal screening conditions which are easy to operate and analyze. Accordingly, we chose the CRISPR library from Zhang Feng’s lab covering 20611 genes in mouse, also designed 1,000 serially uncorrelated sgRNA.Two major parts of the screening system are the screening library and the unique screening method, and the latter is the more important part of the whole screening process. We need to build a screening method which facilitates observation and analysis. According to literature, we chose Clodronate-Liposomes to select for phagocytosis ability. Clodronate was incorporated into liposomes which could been gulped by cell. Once entering the cells, liposomes are degraded by lysosomes resulting in the release of Clodronate, the accumulation of which could lead to the production of ATP-like substances that are toxic to the cells. Theoretically, only macrophages that possess phagocytotic ability can engulf Clodronate-Liposomes, which could induce cell death when its concentration reaches alert threshold. However, cells will survive if the phagocytised genes are regulated to be deleted which lose the phagocytosis ability. Therefore, we can use Clodronate-Liposomes to screen the cells with deleted phagocytic function through cell survival, and identify the key genes which regulate the phagocytic function by aid of the deep sequencing of the cellular genomes.According to the above principles, firstly, a screening system needs tobe established. 1. We need to determine whether Clodronate-Liposomes is specifically phagocytized by the macrophage and its minimum lethal dose. To verify that Clodronate-Liposomes is specifically phagocytized by the macrophage, we introduced Clodronate-Liposomes into iBMM, Hela、L929 and HEK293 cells. Interestingly, only iBMM cells but not Hela、L929 or HEK293 cells exhibited cell death. Using limiting dilution and Trypan blue exclusion to assess cell death, we found that the minimum lethal dose of Clodronate-Liposomes for screening is 700μM. 2. Cells could acquire puromycin resistance following transduction of CRSPR library. Hence the minimum concentration of puromycin needs to be re-determined. Using limiting dilution experiment, we determined the minimum concentration of 1.5μg/m L for puromycin.The other key factor of the screening experiment is to amplify the complete genomic library. According to literature, the conversion rate of the library for the competence E. Coli cells should reach 109cfu/μg to ensure the integrity of library amplification. As a result, we introduced the super competence E. coli, the conversion rate of which is as high as 2×1010 cfu/μg. We also converted the whole-genome library of the CRISPR-treated rat by using the electrically transformed super competence method. According to the calculation based on the number of clones of detection plates in the conversion experiment, the total number of amplified clones were about 3×107,which exceeded the required standard rate(3×106) of complete library amplification and laid a good foundation for the consequent experiments. After library amplification, a virus bearing CRISPR library was implanted in the HEK293 cells. To ensure that only one gene was knocked out in each cell, we detected the titer of the CRISPR library virus in a PI staining method and conducted iBMM cell infection with the virus(MOI = 0.3). After infection, cells were cultured for two weeks with Puro-containing DMEM complete medium before Clodronate-Liposomes was added. Meanwhile, control cells were only treated with Puro-containing medium. After adding Clodronate-Liposomes and culturing for 8 successive days, the survived cells were collected. To further improve the reliability of the screening experiment system, both experimental and control cells were verified at their cytophagy functions with Bioparticle and GFP-E.coli treatment respectively. And the result revealed that the cytophagy function of the cells with the Clodronate-Liposomes drug resistance was reduced remarkably. Furthermore, deep sequencing was applied to the genome extractions from both the experimental and control cells. According to the gene enrichment degree and the software evaluation condition, it was found that the top 100 genes including some genes which have shown to influence the phagocytic ability of macrophages. In addition, the sequencing resμlt also manifested some genes that play important roles in other biological functions. These genes will be the object of our next key research and exploration.In summary, phagocytosis is indispensable for macrophages to exert their immune functions. With Clodronate-Liposomes as the screening reagents and on the basis of the CRISPR rate whole gene library, this research innovatively established the screening system of genes facilitating the regulation on the phagocytic function by the macrophages. This method is easy to use and the screening results could be observed and verified conveniently. For instance, the survived cells may be the cells which could regulate engulfment gene deletion. In addition, through deep sequencing, we screen and obtain the genes influencing the phagocytic function, which lays a foundation for the further exploration on the phagocytosis regulation mechanism of the macrophages, and provides new thoughts and scientific basis for treatment of the diseases caused by phagocytic dysfunction. |
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