相关实验视频

Updated: Jul 9, 2025

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

Published on: March 12, 2018

在CHO细胞系中通过CRISPR-Cas9调解的外部蛋白的稳定表达,通过特定位点的整合

Zhipeng Huang¹, Arslan Habib², Guoping Zhao¹

¹Collaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan University, Shanghai 200438, China.

International journal of molecular sciences

|December 9, 2023

概括

中国仓鼠卵巢细胞现在可以长期稳定地表达外来蛋白质. 克里斯普尔-Cas9技术能够在CHO-K1细胞中实现向基因集成,在许多细胞通道上产生一致的蛋白质.

科学领域:

生物技术是生物技术.
分子生物学分子生物学
细胞生物学细胞生物学

背景情况:

中国仓鼠卵巢 (CHO) 细胞由于其有利的特性,在生物制药生产中被广泛使用.
一个重大挑战是随着时间的推移外源蛋白质表达的下降,这是由随机基因整合到染色体引起的.
治疗性蛋白质的稳定和一致的表达对于生物制造至关重要.

研究的目的:

研究CRISPR-Cas9技术的使用,以有针对性地将外来基因集成到CHO细胞中.
建立一个具有持续外源蛋白表达的CHO细胞系.
为了确定适合稳定基因表达的CHO-K1细胞中的特定染色体位点.

主要方法:

使用CRISPR-Cas9基因编辑技术将外来基因整合到特定的染色体位置.
增强的绿色光蛋白 (EGFP) 和人血清白蛋白 (HSA) 基因是整合的目标.
在CHO-K1细胞中的染色体NC_048595.1上的融合部位被确定为基底434814407附近.
单克隆细胞系培养了60多个通道,以评估表达稳定性.

主要成果:

成功地将EGFP和HSA基因集成到指定的CHO-K1染色体位置 (NC_048595.1).
生成的单克隆细胞系在60多个通道中表现出异源蛋白质的一致表达.
在多个通道中没有观察到表达水平的可辨别变化,这表明高稳定性.

结论:

在CHO-K1染色体NC_048595.1上发现的位置适合稳定,长期的外源蛋白表达.
以CRISPR-Cas9为媒介的有针对性的集成为生物制药生产开发稳定的CHO细胞系提供了强大的战略.
这种方法为创建具有持久和可靠的外源蛋白表达能力的CHO细胞系提供了一种可行的方法.

关键词:

查查查查查查查查查查查这就是CRISPR-Cas9的特征.欧洲粮食计划署 (EGFP) 是一个全球粮食计划署.在HSA中,HSA就是HSA.网站特定的整合.

更多相关视频

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

Published on: August 25, 2018

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Published on: April 20, 2017

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...

Conservative Site-specific Recombination and Phase Variation

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

Homologous Recombination

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...