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在小鼠中使用Cas9 RNA导向核酶进行工程基变化和位标记的等位基

Marina Gertsenstein¹, Lauri G Lintott^1,2, Lauryl M J Nutter^1,2

¹The Centre for Phenogenomics, Toronto, Canada.

Current protocols

|February 25, 2025

概括

本研究详细介绍了一种创建转基因小鼠以研究人类疾病的方法. 研究人员使用CRISPR-Cas9技术在小鼠中引入特定的DNA变化或表位标签,以进行精确的遗传研究.

科学领域:

遗传学和基因组学遗传学和基因组学
分子生物学分子生物学
动物模型动物模型

背景情况:

对于理解疾病因果关系来说,小鼠患者衍生的遗传变化至关重要.
在研究缺乏可用抗体的蛋白质方面,皮标签是必不可少的.
这些基因工程小鼠模型促进了开发,机制和翻译的详细研究.

研究的目的:

用CRISPR-Cas9.9概述一个全面的协议,用于生成使用CRISPR-Cas9.9转基因小鼠.
为疾病研究创建具有特定基变或表位标签的小鼠模型.

主要方法:

用于针对性修改的Cas9原空间子序列的识别.
设计和合成单链寡核酸修复模板和单向导RNA (sgRNAs).
将Cas9,sgRNA微注射或电解,并将修复模板注入小鼠囊中,然后对后代进行查.

主要成果:

成功生成携带精确序列改变或表位标签的小鼠.
实施质量控制措施,以选非目标效应和正确的模板集成.
详细的协议涵盖sgRNA设计,修复模板准备,胚胎操纵和查.

结论:

这种方法提供了一个强大的框架,用于创建定制的鼠标模型.
这些模型对于推进人类疾病的机理学和翻译学研究是非常宝贵的.
描述的协议确保生成高质量的,基因精确的小鼠系.

关键词:

这就是Cas9的情况.电穿孔的电穿孔是什么意思标签标签是表征标签.基因组编辑基因组编辑微注射微注射的方法这里是鼠标鼠标鼠标鼠标鼠标鼠标.一个单核酸变体.

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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.

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...