Construction of IDO1 gene knockout THP-1 cell line using CRISPR/Cas9 and its phenotype identification
Tryptophan metabolism plays an important role in immunometabolism in sepsis,and the expression of indoleamine 2,3-dioxygenase 1(IDO1),the key enzyme of tryptophan metabolism,is up-regulated during sepsis.This study was designed to construct IDO1 gene knockout THP-1 cell line using CRISPR/Cas9,and to provide a cell model for studying the role of IDO1 in macrophage function.Three gRNAs were designed for the IDO1 gene,and inserted into YKO-Lentiviral gRNA plasmid respectively to construct IDO1-gRNA recombinant plasmid,which then confirmed by restriction enzyme digestion and sequencing,and packaged as Lenti-IDO1-gRNA lentivirus.THP-1 cells were infected with Cas9 lentivirus to construct a THP-1 Cas9 cell line stably expressing Cas9 protein.Then THP-1 Cas9 cells were infected with the Lenti-IDO1-gRNA lentivirus for IDO1 gene knockout,and the monoclonal cell lines were screened by limited dilution method.The efficiency of IDO1 knockout was identified by T7E1 digestion test,PCR product sequencing and Western blotting.The proliferative activity of THP-1 cells was detected by CCK8 assay;the expression of CD11b,CD68 and CD 14 in THP-1 macrophages were detected by flow cytometry;and the phagocytic function of THP-1 macrophages was detected by neutral phagocytosis test.T7E1 restriction enzyme digestion results showed that all the three gRNAs could effectively edit IDO1 gene,and gRNA2 had the highest editing efficiency.Sequencing of PCR products showed that IDO1 gene was mutated in three THP-1 IDO 1-KO monoclonal cell lines,and Western blot result showed that IDO1 protein was not expressed,suggesting THP-1 IDO1-KO cells were successfully constructed.CCK-8 proliferation assay showed that IDO1 gene knockout could inhibit the proliferation of THP-1 cells.Flow cytometry showed that IDO1 gene knockout down-regulated the expression of CD1 1b and CD68 in THP-1 macrophages,while up-regulated the expression of CD 14.The results of neutral red phagocytosis test showed that IDO1 gene knockout promoted the phagocytosis of macrophages.In conclusion,the IDO1 gene knockout THP-1 cell line has successfully constructed by CRISPR-Cas9,and IDO1 plays an important role in regulating the proliferation activity and differentiation of THP-1 cells,as well as the phagocytic function of THP-1 macrophages.
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维普
