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[1]郭明睿,胡德庆.利用CRISPR/Cas9技术构建MYC基因的mAID细胞系及其功能验证[J].天津医科大学学报,2026,32(04):350-356.[doi:10.20135/j.issn.1006-8147.2026.04.0350]
　GUO Mingrui,HU Deqing.Construction and functional validation of MYC gene mAID cell line using CRISPR/Cas9 technology[J].Journal of Tianjin Medical University,2026,32(04):350-356.[doi:10.20135/j.issn.1006-8147.2026.04.0350]
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利用CRISPR/Cas9技术构建MYC基因的mAID细胞系及其功能验证(PDF)

《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:
32卷
期数:
2026年04期
页码:
350-356
栏目:
论著
出版日期:
2026-07-10

文章信息/Info

Title:
Construction and functional validation of MYC gene mAID cell line using CRISPR/Cas9 technology
文章编号:
1006-8147（2026）04-0350-07
作者:
郭明睿; 胡德庆
（天津医科大学基础医学院细胞生物学系，天津300070）
Author(s):
GUO Mingrui; HU Deqing
（Department of Cell Biology， School of Basic Medical Sciences，Tianjin Medical University， Tianjin 300070， China）
关键词:
mAID系统; CRISPR/Cas9; HCT116细胞系; MYC
Keywords:
mAID system;  CRISPR/Cas9;  HCT116 cell line;  MYC
分类号:
Q2
DOI:
10.20135/j.issn.1006-8147.2026.04.0350
文献标志码:
A
摘要:
目的：利用改良型生长素诱导降解标签系统，构建能够靶向MYC原癌基因、实现内源性MYC蛋白急性降解的mAID细胞模型。方法：采用CRISPR/Cas9基因编辑技术，设计并构建靶向MYC基因的sgRNA表达载体（PX459-PITCh-MYC-sgRNA）及MYC-mAID同源重组供体质粒。将上述载体共转染至结直肠癌细胞HCT116中，通过药物筛选获得单克隆细胞。利用基因型鉴定与流式细胞术筛选mAID标签正确插入的阳性克隆。通过添加5-Ph-IAA诱导MYC-mAID融合蛋白的降解，并采用蛋白质免疫印迹法验证其降解效率及特异性。结果：成功构建了靶向MYC的sgRNA及mAID供体质粒。经流式细胞术检测证明基因型正确的MYC-mAID阳性克隆荧光信号强度高于WT细胞（n=3，t=123.4，P＜0.000 1）。经流式细胞术检测5-Ph-IAA处理的实验组细胞发现，本系统可快速、特异性诱导内源性MYC-mAID融合蛋白的降解（n=3，F=2 422，P<0.000 1）。结论：成功构建了基于mAID系统的MYC蛋白快速降解细胞系，该模型能够实现内源性MYC蛋白的可控性、急性降解。
Abstract:
Objective： To construct a mAID cell model that can target the MYC oncogene and achieve acute degradation of endogenous MYC protein using an improved auxin-induced degradation tag system. Methods： Using CRISPR/Cas9 gene editing technology， sgRNA expression vectors （PX459-PITCh-MYC-sgRNA） targeting the MYC gene and MYC-mAID homologous recombination plasmids were designed and constructed. These vectors were co-transfected into colorectal cancer cells HCT116， and monoclonal cells were obtained through drug screening. Positive clones with correct mAID tags were screened using genotyping and flow cytometry. 5-Ph-IAA was added to induce the degradation of MYC-mAID fusion protein， and the degradation efficiency and specificity were verified by Western blotting. Results： sgRNA targeting MYC and the mAID donor plasmid were successfully constructed. Flow cytometric analysis confirmed that MYC-mAID-positive clones with the correct genotype exhibited significantly higher fluorescence signal intensity compared to WT cells （n=3， t=123.4， P<0.001）. Furthermore， treatment of the experimental group with 5-Ph-IAA demonstrated that the system could rapidly and specifically induce the degradation of endogenous MYC-mAID fusion proteins （n=3， F=2 422， P<0.000 1）. Conclusion： This study successfully constructs a MYC protein rapid degradation cell system based on the mAID system. This model can achieve controlled and acute degradation of endogenous MYC protein.

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备注/Memo

备注/Memo:
（2026-01-04收稿）
基金项目 国家自然科学基金项目（32270650，32470619）；国家重点研发计划（2024YFA1802300）
作者简介 郭明睿（1998-），女，硕士在读，研究方向：细胞生物学；通信作者：胡德庆，E-mail：hudq@tmu.edu.cn。
更新日期/Last Update: 2026-07-15