相关实验视频

Updated: Jun 19, 2026

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Published on: April 10, 2018

在THP-1细胞和单细胞克隆隔离中以电解基于CRISPR-Cas9为媒介的基因淘汰

Mathieu Pinaud¹, Alessia Zamborlini²

¹INSERM, CEA, Center for Immunology of Viral, Auto-immune, Hematological and Bacterial diseases (IMVA-HB/IDMIT), Université Paris-Saclay.

Journal of visualized experiments : JoVE

|March 17, 2025

概括

这项研究提出了一种高效的CRISPR-Cas9协议,用于工程THP-1细胞,这是人类巨细胞的模型. 该方法实现了高基因淘汰效率,促进了对病毒与宿主相互作用和免疫机制的研究.

科学领域:

细胞生物学细胞生物学
免疫学免疫学免疫学
分子生物学分子生物学

背景情况:

THP-1细胞是人类单细胞衍生的巨细胞广泛使用的模型.
这些细胞模仿初级单细胞,对于研究免疫反应和病原体相互作用至关重要,包括艾滋病毒.
克里斯普尔-Cas9技术为细胞模型中的基因工程提供了一个强大的工具.

研究的目的:

在THP-1细胞中描述CRISPR-Cas9介导基因淘汰的有效协议.
增强免疫相关机制和病毒与宿主相互作用的特征.
优化基因编辑以改善功能研究.

主要方法:

使用了带有电穿孔的CRISPR-Cas9技术,将Cas9:sgRNA核糖蛋白送入THP-1细胞.
采用多个单导向RNA (sgRNAs) 向相同的位置,以诱导大DNA片段的删除.
通过T7内核酶I测定,桑格测序和ICE分析验证了基因编辑;使用免疫和功能测定证实了蛋白质耗尽.

主要成果:

实现高编辑效率,在目标位置达到高达100%的indels.
显示目标蛋白表达的显著下降超过95%.
该协议通过限制稀释来促进单细胞克隆的有效隔离.

结论:

描述的CRISPR-Cas9协议使得THP-1细胞中的高效基因编辑成为可能.
这种优化的方法增强了对巨细胞生物学和宿主-病原体相互作用的研究.
该协议为免疫学研究的髓状细胞模型中的基因操纵提供了一个强大的工具.

更多相关视频

Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

Published on: February 28, 2025

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

Published on: January 10, 2025

相关概念视频

CRISPR

CRISPR

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced Short...

CRISPR/Cas9 Genome Editing

CRISPR/Cas9 Genome Editing

The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...