不稳定EGFP细胞模型的构建及其在基因编辑体系评价中的应用<sup>*</sup>

doi:10.13523/j.cb.2102023

中国生物工程杂志

2021, Vol. 41

Issue (5): 17-26 DOI: 10.13523/j.cb.2102023

技术与方法

不稳定EGFP细胞模型的构建及其在基因编辑体系评价中的应用^*

胡暄^1,²,王松¹,于学玲²,张晓鹏^2,^**(

)

1 国防科技大学新型陶瓷纤维及其复合材料重点实验室长沙 410073
2 军事医学研究院生物工程研究所北京 100071

Construction of a Destabilized EGFP Cell Model for Gene Editing Evaluation

HU Xuan^1,²,WANG Song¹,YU Xue-ling²,ZHANG Xiao-peng^2,^**(

)

1 Science and Technology on Advanced Ceramic Fibers and Composites Laboratory, National University of Defense Technology, Changsha 410073, China
2 Beijing Institute of Biotechnology, Academy of Military Medical Science, Beijing 100071, China

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摘要：

目的:为了更好地评价基因编辑效率,满足高通量筛选应用中快速、高效的检测要求,在细胞上建立一个原位检测方法具有重要的意义。通过检测荧光蛋白信号强度的变化可以评价CRISPR系统在细胞中的基因编辑情况,然而这一方法的效率受限于荧光蛋白较长的半衰期。方法:将鸟氨酸脱羧酶降解结构域(含PEST序列)与EGFP融合,通过慢病毒系统感染HEK-293T细胞,获得了表达单拷贝、EGFP-PEST报告基因的稳转细胞系。结果:与EGFP相比,EGFP-PEST在细胞内的降解速度明显加快,荧光水平在4 h内显著降低。利用该模型比较了3种商品化脂质体介导的CRISPR/Cas9基因编辑效率,能够在2~4 d实现定性和定量评价。结论:这一模型能够快速、灵敏地指示基因编辑效果,可以用于不同CRISPR系统或新递送工具的高通量筛选和评价。

关键词： 荧光蛋白; 单拷贝; CRISPR系统; 细胞模型; 基因编辑

Abstract:

Objective: To meet the requirements of rapid and efficient gene editing detection in high-throughput screening applications, it is of great significance to establish an in situ evaluation method on cells. EGFP can be used to evaluate the gene editing performance mediated by the CRISPR system, but the efficiency is limited by the long half-life of EGFP. Methods: A version of destabilized EGFP (EGFP-PEST) was constructed by fusing degradation domain of ornithine decarboxylase (containing PEST motif) to EGFP. The EGFP-PEST gene was introduced into the chromosome of HEK-293T cells by lentivirus, and the copy number of EGFP-PEST gene was measured by RT-qPCR. Finally, cell strains with single copy of EGFP-PEST transgene were established. Results: Compared to unmodified EGFP, the cellular fluorescence of EGFP-PEST significantly decreased within 4 h, suggesting efficient PEST-mediated protein degradation. The cell model was used to evaluate the potential of three commercial lipids to deliver CRISPR/Cas9 complex. Data showed that gene editing was detected in 2-4 days by quantitative or qualitative measurements. Conclusion: This cell model can be used in high-throughput screening for new CRISPR tools or novel delivery systems by indicating the gene editing rapidly and sensitively.

Key words: Fluorescent protein Single copy CRISPR system Cell model Gene editing

收稿日期: 2021-02-22 出版日期: 2021-06-01

ZTFLH:

Q812

基金资助: * 国家自然科学基金(81703048)

通讯作者: 张晓鹏 E-mail: zxp8565@aliyun.com

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引用本文:

胡暄,王松,于学玲,张晓鹏. 不稳定EGFP细胞模型的构建及其在基因编辑体系评价中的应用^*[J]. 中国生物工程杂志, 2021, 41(5): 17-26.

HU Xuan,WANG Song,YU Xue-ling,ZHANG Xiao-peng. Construction of a Destabilized EGFP Cell Model for Gene Editing Evaluation. China Biotechnology, 2021, 41(5): 17-26.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2102023 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I5/17

图1 pLVX-EGFP-PEST质粒构建的原理示意图

图2 HEK-293T细胞活性与Puromycin抗性浓度的关系

图3 pLVX-EGFP-PEST质粒构建

图4 HEK-293T细胞的荧光细胞比例与感染时病毒原液体积的关系

图5 基于泊松分布模拟病毒感染细胞的数学模型

图6 绝对定量PCR 标准品扩增图谱、标准曲线拟合图和熔解曲线分析图

表1 RT-qPCR方法获得的细胞EGFP-PEST基因拷贝数

图7 流式分析各细胞株的荧光表达情况

图8 EGFP-PEST(a)和EGFP(b)在CHX处理的293T细胞中的荧光信号变化

图9 jetCRISPR递送Cas9/sgRNA进293T. EGFP-PEST细胞的荧光表达

图10 RNAiMAX和Lipo2000转导Cas9/sgRNA 4 d后293T.EGFP-PEST细胞的荧光表达

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