应用CRISPR-Cas9技术构建RIG-I基因敲除的HEK293细胞系

Knockout of RIG-I in HEK293 cells by CRISPR/Cas9

摘要:

为了揭示维甲酸诱导基因I (retinoic acid inducible-gene I, RIG-I)基因敲除对I型干扰素信号通路中关键因子的影响，本研究利用CRISPR/Cas9技术构建了RIG-I基因敲除的HEK293细胞。首先设计了3条针对靶标基因的单向导RNA (single guide RNA, sgRNA)，并构建了pX459重组载体。重组载体转染HEK293细胞后，通过嘌呤霉素筛选细胞株，再以聚肌苷酸-聚胞苷酸(polyinosinic acid-polycytidylic acid, poly I:C)为病毒模拟物转染细胞，通过基因测序、荧光定量PCR、免疫印迹和免疫荧光的方法检测RIG-I基因的敲除情况，同时对I型干扰素信号通路中的黑色素瘤分化相关基因5 (melanoma differentiation-associated protein 5, MDA5)、干扰素β1 (interferon β1, IFNβ1)和核转录因子kappa B p65[nuclear transcription factor kappa B p65, NF-κb(p65)]等关键因子以及细胞活力进行分析。结果表明，本研究成功构建了2株稳定敲除RIG-I基因的HEK293细胞株(S1和S3)，RIG-I在S1和S3中的基因转录水平和蛋白质表达水平均显著低于野生型细胞(P<0.05)。S1和S3细胞株的MDA5和IFNβ1基因转录水平和S3细胞株的NF-κB(p65)蛋白质水平显著低于野生细胞株(P<0.05)；细胞免疫荧光分析结果表明，poly I:C转染细胞后，与野生型相比，S1细胞株核外存在较多的NF-κB(p65)蛋白。此外，poly I:C转染细胞48 h后，显著降低了野生型和S1细胞株的活力(P<0.05)，但是不影响S3细胞株的活力。综上所述，本实验通过CRISPR/Cas9系统成功构建了2株RIG-I基因敲除的HEK293细胞，为进一步研究I型干扰素信号通路的机制提供了稳定的细胞模型。

Abstract:

We knocked out the retinoic acid-inducible gene I (RIG-I) in HEK293 cells via CRISPR/Cas9 to reveal the effects of RIG-I knockout on the key factors in the type I interferon signaling pathway. Three single guide RNAs (sgRNAs) targeting RIG-I were designed, and the recombination vectors were constructed on the basis of the pX459 vector and used to transfect HEK293 cells, which were screened by puromycin subsequently. Furthermore, a mimic of virus, poly I:C, was used to transfect the cells screened out. RIG-I knockout was checked by sequencing, real-time quantitative PCR, Western blotting, and immunofluorescence assay. Meanwhile, the expression levels of key factors of type I interferon signaling pathway such as melanoma differentiation-associated gene 5 (MDA5), interferonβ1 (IFNβ1), and nuclear factor-kappa B p65 [NF-κB(p65)], as well as cell viability, were determined. The results showed that two HEK293 cell lines (S1 and S3) with RIG-I knockout were obtained, which exhibited lower mRNA and protein levels of RIG-I than the wild type HEK293 cells (P<0.05). The mRNA levels of MDA5 and IFNβ1 in S1 and S3 cells and the protein level of NF-κB(p65) in S3 cells were lower than those in the wild type (P<0.05). More extranuclear NF-κB(p65) protein was detected in S1 cells than in the wild type after transfection with poly I:C. Plus, the wild-type and S1 cells transfected with poly I:C for 48 h showcased reduced viability (P<0.05), while S3 cells did not display the reduction in cell viability. In summary, the present study obtained two HEK293 cell lines with RIG-I knockout via CRISPR/Cas9, which provided a stable cell model for exploring the mechanism of type I interferon signaling pathway.