[1]王佩佩,章笑甜,凌志明,等.EGFP报告基因PiggyBac载体在三阴性乳腺癌MDA-MB-231细胞系电转染条件优化[J].现代检验医学杂志,2025,40(03):189-194.[doi:10.3969/j.issn.1671-7414.2025.03.035]
　WANG Peipei,ZHANG Xiaotian,LING Zhiming,et al.Optimization of Electrotransfection Conditions of EGFP Reporter Gene PiggyBac Vector in Triple Negative Breast Cancer MDA-MB-231 Cell Line[J].Journal of Modern Laboratory Medicine,2025,40(03):189-194.[doi:10.3969/j.issn.1671-7414.2025.03.035]
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EGFP报告基因PiggyBac载体在三阴性乳腺癌MDA-MB-231细胞系电转染条件优化()

《现代检验医学杂志》[ISSN:/CN:]

卷:
第40卷
期数:
2025年03期
页码:
189-194
栏目:
研究简报·实验技术
出版日期:
2025-05-15

文章信息/Info

Title:
Optimization of Electrotransfection Conditions of EGFP Reporter Gene PiggyBac Vector in Triple Negative Breast Cancer MDA-MB-231 Cell Line
文章编号:
1671-7414（2025）03-189-06
作者:
王佩佩1,2，章笑甜1，凌志明1，王文娟1，刘秀盈1，王建勋1,3
（1.北京中医药大学生命科学学院，北京 102488；2.中国医学科学院肿瘤医院山西医院，太原 030000；3.深圳北京中医药大学研究院，广东深圳 518118）
Author(s):
WANG Peipei1,2，ZHANG Xiaotian1，LING Zhiming1，WANG Wenjuan1，LIU Xiuying1，WANG Jianxun1,3
(1.School of Life Sciences，Beijing University of Chinese Medicine，Beijing 102488，China；2.Shanxi Hospital , Cancer Hospital of Chinese Academy of Medical Sciences，Taiyuan 030000, China；3.Shenzhen Research Institute, Beijing University of Chinese Medicine，Guangdong Shenzhen 518118, China)
关键词:
三阴性乳腺癌; 电转染; MDA-MB-231; PiggyBac转座子
分类号:
R737.9；R730.43
DOI:
10.3969/j.issn.1671-7414.2025.03.035
文献标志码:
A
摘要:
目的设置不同电转染条件，探讨含增强型绿色荧光蛋白（EGFP）报告基因稳定转入人乳腺癌细胞MDA-MB-231细胞的优化条件。方法构建含EGFP报告基因的PiggyBac（PB）转座子系统，并控制电转染MDA-MB-231细胞时的波形、电压、电击时间、电击次数、质粒浓度、细胞密度、转座子与转座酶比例等转染条件，采用流式细胞术荧光异硫氰酸荧光素（FITC）通道检测其转染效率，并于激光扫描共聚焦显微镜下观察荧光蛋白表达情况，验证EGFP报告基因电转染效率与表达情况，分析不同电转染条件下MDA-MB-231细胞的电转效率。结果MDA-MB-231细胞的电转染优化条件为电压280V，波形为指数波，电击1次，转座子质粒浓度1000ng/μl左右，转座子比转座酶质量比例1:1，细胞数量为2×106个，电转染率可达60.23%±5.63%，且MDA-MB-231细胞状态良好。结论成功优化MDA-MB-231细胞的电转染条件，达到稳定高效转染，为MDA-MB-231细胞株的相关基础研究提供高效电转实验参数。
Abstract:
Objective To explore the optimal conditions for stable transfer of reporter gene containing enhanced green fluorescent protein (EGFP) into human breast cancer cells MDA-MB-231 cells under different electrotransfection conditions. Methods A PiggyBac（PB）transposon system containing EGFP reporter gene was constructed, and transfection conditions such as waveform, voltage, shock time, shock times, plasmid concentration, cell density, transposon to transposase ratio were controlled during electrotransfection of MDA-MB-231 cells. The transfection efficiency was detected by flow cytometry fluorescein isothiocyanate（FITC）channel. The expression of fluorescent protein was observed under laser scanning confocal microscope, the electrotransfection efficiency and expression of EGFP reporter gene were verified, and the electrotransfer efficiency of MDA-MB-231 cells was analyzed under different electrotransfection conditions. Results The optimal transfection conditions of MDA-MB-231 cells were as follows: voltage 280V, exponential wave, electric shock once, concentration of transposon plasmid about 1 000 ng/μl, mass ratio of transposon to transpotase 1:1, number of cells 2×106, electrotransfection rate up to 60.23%±5.63%. The state of MDA-MB-231 cells was good. Conclusion The electrotransfection conditions of MDA-MB-231 cells were successfully optimized to achieve stable and efficient transfection, which provided efficient electrotransfer experimental parameters for the relevant basic research of MDA-MB-231 cell line.

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备注/Memo

备注/Memo:
基金项目：北京中医药大学高层次人才科研启动经费项目（批准号：9011451310032）。
作者简介： 王佩佩（1993-），女，博士，研究员，研究方向：肿瘤免疫治疗，E-mail:1974333166@qq.com。章笑甜（1999-），女，硕士研究生，研究方向：肿瘤免疫治疗，E-mail:zhangxiaotian0821@163.com。共同第一作者。
通讯作者：王建勋（1973-），男，美籍，特聘教授，博士生导师，研究方向：肿瘤免疫治疗，E-mail:jianxun.wang@bu
更新日期/Last Update: 2025-05-15