利用CRISPR/Cas9技术构建<i>TRIM21</i>敲除稳定HeLa细胞系

doi:10.7668/hbnxb.20195539

摘要/Abstract

摘要：

旨在利用CRISPR/Cas9基因编辑技术,成功构建TRIM21基因敲除的稳定人宫颈癌细胞(HeLa)系。首先,针对TRIM21基因设计并合成了2个特异性的sgRNA(sgRNA1和sgRNA2),通过PCR扩增和无缝克隆技术将其分别克隆至LentiCRISPRv2载体中。随后,利用电转染法将编辑载体转染至HeLa细胞中,并通过嘌呤霉素筛选获得成功整合编辑载体的单克隆细胞系。经Western Blot验证和扩大培养,获得了稳定敲除TRIM21的HeLa细胞系。基于该细胞系,进一步探究了TRIM21在调控Ⅰ型干扰素信号通路中的作用。试验结果表明,TRIM21敲除显著抑制了细胞内MDA5、MAVS和IRF3的表达水平,同时抑制了IRF3的磷酸化,揭示了TRIM21在Ⅰ型干扰素信号通路中的重要调控作用。成功构建的TRIM21敲除HeLa细胞系为深入研究TRIM21调控病毒增殖机制及其在先天免疫应答中的作用提供了可靠的细胞模型;通过优化试验方法,证实了电转染法在构建基因敲除细胞系中的高效性和稳定性,为其他基因敲除细胞系的构建提供了技术参考。

关键词: 细胞系, TRIM21, CRISPR/Cas9, 电转染, 人宫颈癌细胞HeLa

Abstract:

It aimed to successfully construct a stable human cervical cancer (HeLa) cell line with TRIM21 gene knockout using CRISPR/Cas9 gene editing technology.First,two specific sgRNAs (sgRNA1 and sgRNA2) targeting the TRIM21 gene were designed and synthesized,and they were cloned into the LentiCRISPRv2 vector using PCR amplification and seamless cloning techniques.Subsequently,the editing vectors were transfected into HeLa cells via electroporation,and monoclonal cell lines with successfully integrated editing vectors were obtained through puromycin screening.After validation by Western Blot and expansion of culture,a stable TRIM21-knockout HeLa cell line was established.Based on this cell line,we further investigated the role of TRIM21 in regulating the type I interferon signaling pathway.The experimental results demonstrated that TRIM21 knockout significantly suppressed the expression levels of MDA5,MAVS,and IRF3 in the cells,while also inhibiting the phosphorylation of IRF3,revealing the critical regulatory role of TRIM21 in the type I interferon signaling pathway.The TRIM21-knockout HeLa cell line successfully constructed in this study provides a reliable cellular model for in-depth research into the mechanism of TRIM21 in regulating viral replication and its role in innate immune responses.Furthermore,by optimizing experimental methods,this study confirmed the efficiency and stability of electroporation in constructing gene-knockout cell lines,offering technical references for the construction of other gene-knockout cell lines.

Key words: Cell line, TRIM21, CRISPR/Cas9, Electroporation, Human cervical cancer cell (HeLa)

中图分类号:

S813.3生物技术遗传育种

杨东亮, 邵帅, 纪晓岚, 张德荣, 柏家林, 李琼毅. 利用CRISPR/Cas9技术构建TRIM21敲除稳定HeLa细胞系[J]. 华北农学报, 2025, 40(6): 231-238. doi: 10.7668/hbnxb.20195539.

YANG Dongliang, SHAO Shuai, JI Xiaolan, ZHANG Derong, BAI Jialin, LI Qiongyi. Construction of TRIM21 Knockout Stable Hela Cell Line by CRISPR/Cas9 Technology[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(6): 231-238. doi: 10.7668/hbnxb.20195539.

http://www.hbnxb.net/CN/Y2025/V40/I6/231

图/表 5

图1 TRIM21-Cas9-sgRNA-LentiCRISPRv2 质粒序列 A.重组TRIM21-Cas9-sgRNA1-LentiCRISPRv2 质粒序列;B.重组TRIM21-Cas9-sgRNA2-LentiCRISPRv2 质粒序列。

Fig.1 Sequence of TRIM21-Cas9-sgRNA-LentiCRISPRv2 plasmid A.Sequence of the recombinant TRIM21-Cas9-sgRNA1-LentiCRISPRv2 plasmid; B.Sequence of the recombinant TRIM21-Cas9-sgRNA2-LentiCRISPRv2 plasmid.

图2 PCR产物电泳图 A.sgRNA1 PCR扩增产物(M.DL5000 DNA Marker);B.sgRNA2 PCR扩增产物(M.DL5000 DNA Marker)。

Fig.2 Electrophoresis image of PCR products A.PCR amplification product of sgRNA1 (M.DL5000 DNA Marker);B.PCR amplification product of sgRNA2 (M.DL5000 DNA Marker).

图3 TRIM21-sgRNA质粒双酶切验证 A.TRIM21-sgRNA1质粒经Kpn Ⅰ和EcoR Ⅴ双酶切验证(M.1 kb DNA Marker);B.TRIM21-sgRNA2质粒经Kpn Ⅰ和EcoR Ⅴ双酶切验证(M.1 kb DNA Marker)。

Fig.3 TRIM21-sgRNA plasmid double enzyme digestion verification A.Verification of TRIM21-sgRNA1 plasmid by double digestion with Kpn Ⅰ and EcoR Ⅴ(M.1 kb DNA Marker);B.Verification of TRIM21-sgRNA2 plasmid by double digestion with Kpn Ⅰ and EcoR Ⅴ(M.1 kb DNA Marker).

图4 TRIM21敲除验证 A.筛选后5个TRIM21敲除单克隆细胞(1—5)及1个阴性对照单克隆细胞;B.Western Blot检测5个TRIM21敲除单克隆细胞中TRIM21表达水平;C.TRIM21 KO#1细胞系、TRIM21 NC细胞系和HeLa细胞系形态。比例尺.100 μm。

Fig.4 TRIM21 knockout verification A.Five TRIM21 knockout monoclonal cell lines (1—5) post-screening and one negative control monoclonal cell line;B.Western Blot analysis of TRIM21 expression levels in the five TRIM21 knockout monoclonal cell lines;C.Morphology of the TRIM21 KO#1 cell line,TRIM21 NC cell line,and HeLa cell line.Scale bar.100 μm.

图5 敲除TRIM21功能验证 A.Western Blot检测TRIM21敲除后Ⅰ型干扰素信号通路中信号分子MDA5、MAVS、IRF3及p-IRF3表达情况,其中NC即表示提取TRIM21 NC细胞系总蛋白,KO即表示提取TRIM21 KO#1细胞系总蛋白;B—F.对TRIM21、MDA5、MAVS、IRF3及p-IRF3进行灰度分析,不同字母表示差异显著(P<0.001)。

Fig.5 TRIM21 knockout functional verification A.Western Blot was used to detect the expression of signal molecules MDA5,MAVS,IRF3 and p-IRF3 in type Ⅰ interferon signaling pathway after TRIM21 knockout,NC was used to extract the total protein of TRIM21 NC cell line,and KO was used to extract the total protein of TRIM21 KO#1 cell line;B—F.Gray analysis of TRIM21,MDA5,MAVS,IRF3 and p-IRF3,different letters indicate significant difference(P<0.001).

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利用CRISPR/Cas9技术构建TRIM21敲除稳定HeLa细胞系

Construction of TRIM21 Knockout Stable Hela Cell Line by CRISPR/Cas9 Technology

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