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

卷:

31卷

期数:

2025年03期

页码:

231-236,250

栏目:

基础医学

出版日期:

2025-05-20

文章信息/Info

Title:

Establishment and functional verification of mouse embryonic stem cell dTAG cell line targeting SUPT6

文章编号:

1006-8147(2025)03-0231-07

作者:

于艺晞; 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):

YU Yixi; HU Deqing

（Department of Cell Biology，School of Basic Medical Sciences，Tianjin Medical University，Tianjin 300070，China）

关键词:

SUPT6; CRISPR/Cas9; dTAG; 胚胎干细胞

Keywords:

SUPT6; CRISPR/Cas9; dTAG; embryonic stem cells

分类号:

Q2

DOI:

10.20135/j.issn.1006-8147.2025.03.0231

文献标志码:

A

摘要:

目的：利用降解TAG系统（dTAG）建立靶向SPT6同系物、组蛋白伴侣和转录延伸因子（SUPT6）的dTAG细胞系，诱导内源性SUPT6的急性降解。方法：设计靶向小鼠Supt6的向导RNA并构建PX459-PITCh-Supt6 sgRNA和Supt6-dTAG供体质粒；将载体转入小鼠胚胎干细胞V6.5细胞系中并进行药物筛选，对筛选后的单克隆进行基因型鉴定及测序分析；CCK-8法及碱性磷酸酶染色检测阳性克隆的增殖与分化；使用dTAG-13诱导阳性克隆SUPT6蛋白的降解并进行免疫印迹实验验证；碱性磷酸酶染色评估阳性克隆中SUPT6降解对干细胞多能性的影响。结果：成功设计并构建靶向Supt6向导RNA和dTAG供体载体；筛选获得插入dTAG标签的阳性克隆；插入dTAG标签不影响细胞的增殖与分化功能（F=0.014 19，P＞0.05）；dTAG-13成功诱导Flag标签标记的内源性SUPT6降解（F=617.5、372.4，均P＜0.001），且撤药后Flag标签标记的SUPT6水平逐渐恢复（F=410.9、226.8，均P＜0.001）；阳性克隆中SUPT6降解导致干细胞多能性下降。结论：构建的SUPT6-dTAG细胞系能够实现内源性SUPT6的急性可逆降解。

Abstract:

Objective： A dTAG cell line targeting SPT6 homologous，histone chaperone and transcription extension factor （SUPT6） was established using the degradation TAG（dTAG） system to induce acute degradation of endogenous SUPT6. Methods：The small guide RNA targeting mouse Supt6 was designed，and PX459-PITCh-Supt6 sgRNA and Supt6-dTAG donor plasmids were constructed.These plasmids were co-transfected into the mouse embryonic stem cell line V6.5 followed by drug selection.The selected monoclones were genotypically identified and sequenced.The proliferation and differentiation of positive clones were detected by CCK-8 assay and alkaline phosphatase staining. The degradation of SUPT6 in positive clones was induced by dTAG-13 and confirmed by Western blotting. Alkaline phosphatase staining was used to evaluate the effect of SUPT6 degradation on stem cell pluripotency in positive clones. Results： Targeted Supt6 guide RNA and dTAG donor vectors were successfully designed and constructed. Positive clones with the dTAG tag inserted were obtained. The insertion of the dTAG tag did not affect cell proliferation and differentiation（F=0.014 19，P＞0.05）. dTAG-13 effectively induced degradation of endogenous Flag-tagged SUPT6（F=617.5，372.4，both P<0.001）. Upon drug withdraw-al，the levels of Flag-tagged SUPT6 gradually recovered（F=410.9，226.8，both P<0.001）. SUPT6 degradation in positive clones led to a reduction in stem cell pluripotency. Conclusion：The constructed SUPT6-dTAG cell line is capable of achieving acute and reversible degradation of endogenous SUPT6.

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备注/Memo

备注/Memo:

基金项目: 国家自然科学基金面上项目（32270650）
作者简介: 于艺晞（1998-），女，硕士在读，研究方向：医学细胞生物学；
通信作者：胡德庆，E-mail：hudq@tmu.edu.cn。

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更新日期/Last Update: 2025-06-01