稳定表达敲入增强绿色荧光蛋白标记的干扰素γ受体2基因Ifngr2的黑素瘤细胞系的构建与鉴定

Construction and identification of B16 cell line stably expressing EGFP-labeled interferon-γreceptor 2 gene Ifngr2

目的构建稳定表达敲入增强绿色荧光蛋白(EGFP)标记的干扰素γ(IFN-γ)受体2基因Ifngr2的黑素瘤B16细胞系,进行IFNGR2表达和功能调控研究。方法首先应用http://chopchop.cbu.uib.no/网站设计引导RNA,使用分子克隆的方法构建基于CRISPR-Cas9和CRIS-PITCh(v2)的表达载体,并将2种载体使用LTX共同转染入B16细胞系,筛选单克隆。然后,应用基因组PCR、流式分析以及荧光显微镜技术,对基因敲入位置和基因表达进行分析鉴定。对阳性克隆E10和B4进行IFN-γ20 mg·L-1刺激48 h,分析IFN-γ下游靶基因的表达以检测阳性细胞系对IFN-γ的反应性。最后应用流式分析和实时荧光定量PCR(RT-PCR)检测肿瘤坏死因子α(TNF-α)20 mg·L-1和奥沙利铂2 mmol·L-1处理3和48 h后,E10和B4细胞系Ifngr2和EGFP的表达。结果基因组PCR及测序分析显示,PCR产物与预期一致,EGFP插入基因组正确位置。流式细胞分析及荧光显微镜观察分析,可看到绿色荧光峰值右移及绿色荧光,说明该细胞系正常表达EGFP。RT-PCR分析显示,IFN-γ刺激后,Cd274(PDL1)在阳性克隆E10和B4细胞中显著上调63和138倍,Psmb9显著上调2885和4616倍,上调幅度与B16母细胞相当,说明E10和B4细胞系具有正常的IFN-γ信号通路。流式细胞分析显示,TNF-α处理后,E10和B4细胞平均荧光强度由3845和4456上升到5720和5972;奥沙利铂处理后,E10和B4细胞平均荧光强度由3032和2786上升到4285和4179。RT-PCR分析显示,TNF-α刺激后,Ifngr2在B16,E10和B4细胞中分别上升11.40,9.31和21.39倍,EGFP在E10和B4细胞中分别上升2.50和5.37倍;奥沙利铂刺激后,Ifngr2在B16,E10和B4细胞中分别上升10.99,3.54和5.39倍,EGFP在E10和B4细胞中分别上升2.70和2.44倍。IFN-γ处理不能上调Ifngr2和EGFP的表达,因此绿色荧光强度反映了Ifngr2的表达。结论成功构建稳定敲入EGFP标记的Ifngr2基因的黑素瘤细胞系,发现TNF-α和奥沙利铂可上调Ifngr2和EGFP的表达,表明该细胞系可用于IFNGR2表达和功能研究。

OBJECTIVE To construct a melanoma B16 cell line stably expressing the EGFP-labeled interferon-γreceptor 2 gene Ifngr2,and to study the expression and function regulation of IFNGR2.METHODS Firstly,Ifngr2 guide RNA was designed using the website http://chopchop.cbu.uib.no/,and a molecular cloning method was used to construct an expression vector based on CRISPR-Cas9 and CRIS-PITCh(v2).Both vectors were co-transfected into B16 cell lines with LTX and screened for monoclonals.Then,the genomic PCR,flow cytometry and fluorescence microscope observation techniques were used to identify the clones,and the gene knock-in position and gene expression were identified and analyzed.Positive clones E10 and B4 were stimulated with interferon-γ(IFN-γ)20μg·L-1 for 48 h and analyzed for IFN-γdownstream target gene expression to detect the positive cell line′s reactivity to IFN-γ.Finally,flow cytometry and real-time quantitative PCR were used to detect the expressions of Ifngr2 and EGFP after stimulation with tumor necrosis factor-α(TNF-α)(20μg·L-1)and oxaliplatin(2 mmol·L-1)for 3 h and 48 h respectively in positive clones.RESULTS Genomic PCR analysis showed that the expected product was obtained,and EGFP was inserted into the correct position in the genome.Flow cytometric analysis and fluorescence microscopic observation analysis showed that the green fluorescence peak shifted to the right and green fluorescence could be seen,indicating that the cell lines normally expressed EGFP.Real-time fluorescence quantitative PCR analysis showed that after IFN-γstimulation,Cd274(PDL1)was significantly upregulated by 63,138 times in E10 and B4 cells,and Psmb9 was significantly upregulated by 2885 and 4616 times in E10 and B4 cells,indicating that E10 and B4 cell lines had normal IFN-γsignaling pathway.Flow cytometry analysis showed that after treatment with TNF-αand oxaliplatin,the peak of green fluorescence in E10 and B4 cell lines shifted to the right.After TNF-αtreatment,the average fluorescence intensity of E10 and B4 increased from 3845 and 4456 to 5720 and 5972.After oxaliplatin treatment,the average fluorescence intensity of E10 and B4 increased from 3032 and 2786 to 4285 and 4179.Real-time fluorescence quantitative PCR analysis showed that after TNF-αstimulation,Ifngr2 increased by 11.40,9.31 and 21.39 times in B16,E10,and B4 cells,respectively,and EGFP increased by 2.50 and 5.37 times in E10 and B4 cells,respectively.After oxaliplatin stimulation,Ifngr2 increased by 10.99,3.54,and 5.39 times in B16,E10,and B4 cells,respectively,and EGFP increased by 2.70 and 2.44 times in E10 and B4 cells,respectively.IFN-γtreatment could not upregu⁃late the expression of Ifngr2 or EGFP,so the intensity of green fluorescence reflected the expression of Ifngr2.CONCLUSION The melanoma B16 cell line stably knocking in the green fluorescent protein EGFP-labeled Ifngr2 gene is successfully established.It is found that TNF-αand oxaliplatin can up-regulate the expressions of Ifngr2 and EGFP,indicating that the cell line can be used for IFNGR2 expression and function research.