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一步一步的协议,使一个Knock-in Xenopus laevis可视化内源性基因表达

Norie Kagawa¹, Yoshihiko Umesono¹, Ken-Ichi T Suzuki²

¹Department of Life Science, Graduate School of Science, University of Hyogo, Akougun, Hyogo, Japan.

Development, growth & differentiation

|June 9, 2025

概括

研究人员为Xenopus laevis开发了一种名为NEXTi的新CRISPR-Cas9技术,使得精确的记者基因敲击能够研究内生基因表达模式. 这种方法可以通过特定组织的eGFP表达来跟踪基因活性.

科学领域:

分子生物学分子生物学
发展生物学发展生物学
遗传学是一个遗传学.

背景情况:

研究Xenopus laevis的内源基因表达对于理解发育过程至关重要.
现有的方法可能缺乏效率或回顾本地监管元素的能力.
记者基因表达是可视化基因活动的强大工具.

研究的目的:

建立和验证一种新的,高效的对Xenopus laevis的敲入技术.
用记者基因使内源基因表达模式的复制成为可能.
为这种新技术的应用提供详细的协议.

主要方法:

开发一种基于CRISPR-Cas9的敲门策略,命名为NEXTi (新的和简单的Xenopus有针对性的集成).
将捐赠者DNA与eGFP记者基因集成到目标基因的5'未翻译区域 (UTR).
在Xenopus胚胎中微注射成分,然后进行组织特异性表达分析和育种以确认F1代.

主要成果:

成功生成了具有组织特异性EGFP表达 (2-13%效率) 的敲入Xenopus胚胎.
确认了针对特定位点 (krt.12.2.L,myod1.S,sox2.L,bcan.S) 的有针对性的整合.
在F1代胚胎中,从注射成年青中获得稳定的eGFP表达.

结论:

NEXTi技术提供了一种有效的方法,用于生成具有反映内源基因调节的记者基因表达的Xenopus模型.
这种技术有助于研究Xenopus在发育过程中的基因功能和调节.
描述的协议允许可重复生成和验证敲进线.

关键词:

它们是 Xenopus laevis 的类型.5′未翻译的区域克里斯普尔-卡斯9是什么意思敲门进入敲门进入

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相关概念视频

In-vitro Mutagenesis

In-vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.