基于CRISPR/Cas9系统建立GPR108缺失的THP-1细胞株及其功能初步研究

Construction of THP-1 cell line with GPR108 gene deletion by CRISPR/Cas9 system and exploration of its function

目的建立稳定敲除G蛋白偶联受体108(GPR108)基因的人单核细胞白血病细胞系(THP-1),并初步研究其功能。方法根据人GPR108基因序列设计,合成可应用于成簇有规律的间隔短回文重复序列及其相关蛋白9(CRISPR/Cas9)的单链引导RNA(sgRNA1和sgRNA2)对应的DNA,利用分子克隆技术在pL-CRISPR.EFS.GFP慢病毒载体中插入sgRNA1和sgRNA2序列,构建重组载体(pL-CRISPR.EFS.GFP-sgRNA1和pL-CRISPR.EFS.GFP-sgRNA2)。将测序正确的重组体与包装质粒(pMD2.G和psPAX2)共转染293T细胞进行病毒包装,收集病毒液并感染THP-1细胞。利用流式细胞分选技术分离GFP+细胞于96孔培养板中,培养获得单细胞克隆。利用聚合酶链式反应(PCR)和蛋白免疫印迹法(Western blot)检测THP-1细胞中GPR108是否敲除成功。应用脂多糖(LPS)刺激GPR108-/-和GPR108+/+的THP-1细胞,Western blot法检测细胞内白细胞介素-8(IL-8)的表达,流式微球技术(CBA)检测细胞培养上清液中的IL-8浓度。结果成功构建重组慢病毒载体pL-CRISPR.EFS.GFP-sgRNA,转染THP-1细胞后通过流式细胞仪分选获得单细胞克隆F9。PCR和Western blot法均证实F9是GPR108-/-THP-1单细胞克隆。LPS刺激GPR108-/-和GPR108+/+THP-1细胞,Western blot和CBA的结果均显示敲除GPR108的THP-1细胞中IL-8的合成和分泌明显减少。结论成功建立GPR108缺失的THP-1细胞株;缺失GPR108的THP-1细胞在受到LPS刺激时产生的趋化因子IL-8显著降低。为后续研究GPR108在免疫炎症中的作用奠定了基础。

Objective To establish a Tohoku hospital pediatrics-1(THP-1)cell line with G protein-coupled receptor108(GPR108)deletion and explore its functions.Methods According to the requirements of the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system,two single guide RNAs(sgRNA1 and sgRNA2)paring to the flanking fragments of human GPR108 gene were designed and synthesized.The two oligonucleotides were inserted in the pL-CRISPR.EFS.GFP vector to generate the new recombinant vectors(pL-CRISPR.EFS.GFP-sgRNA1 and pL-CRISPR.EFS.GFP-sgRNA2).The recombinant vectors and packaging plasmids(pMD2.G and psPAX2),were co-transfected into 293T cells to generate virus for infecting THP-1 cells.The GFP+cells were screened and isolated in 96-well culture plates by flow cytometry to obtain single-cell clones.PCR and Western blot were used to detect whether GPR108 was successfully knocked out in THP-1 cells.Both GPR108+/+and GPR108-/-THP-1 cells were treated with lipopolysaccharide(LPS).Interleukin 8(IL-8)derived from the THP-1 cells,which were treated by LPS,was detected with Western blot and cytometric bead array(CBA)analysis.Results The recombinant lentiviral vector pL-CRISPR.EFS.GFP-sgRNA was successfully constructed and single-cell clone F9 was obtained by flow cytometric sorting after transfection of THP-1 cells.PCR and Western blot both confirmed that F9 was a GPR108-/-THP-1 single-cell clone.LPS stimulated GPR108-/-and GPR108+/+THP-1 cells,both Western blot and CBA results showed a significant decrease in IL-8 synthesis and secretion in GPR108-/-THP-1 cells.Conclusion The GPR108-/-THP-1 cell clone is successfully obtained based on the CRISPR/Cas9 system.GPR108 deletion in THP-1 cells treated by LPS leads to a decrease of IL-8 expression and secretion.It lays the foundation for further research on the molecular mechanisms of GPR108 in the immune inflammatory response.