通过 CRISPR/Cas9 介导的基因靶向技术生成条件性 Acvrl1 敲除小鼠。

Generation of conditional Acvrl1 knockout mice by CRISPR/Cas9-mediated gene targeting.

机构信息

Department of Anesthesiology, Huashan Hospital, Fudan University, Shanghai 200040, China.

Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai 200040, China.

出版信息

Mol Cell Probes. 2018 Feb;37:32-38. doi: 10.1016/j.mcp.2017.11.003. Epub 2017 Nov 10.

DOI:10.1016/j.mcp.2017.11.003

PMID:29129659

Abstract

OBJECTIVES

This study aimed to generate mutant mice containing the Acvrl1 gene flanked with LoxP sequences to allow conditional deletion of Acvrl1 by the LoxP/Cre system. Such mice may facilitate the development of brain arteriovenous malformation (BAVM) models.

METHODS

The CRISPR/Cas9 technique was used to edit Acvrl1. Two single guide RNAs (sgRNAs) with recognition sites on intron 3 and 8 and a donor vector that was homologous with the targeted gene and contained two LoxP sequences were designed and constructed. The in vitro-synthesized sgRNA, Cas9 mRNA and donor vectors were injected into mouse zygotes, which were then transferred into pseudopregnant mice. Neonatal mutant mice were identified by genotyping and sequencing.

RESULTS

Two mice with a floxed Acvrl1 allele were generated at a success rate of 8.7%. The target mice, which were healthy and fertile, were obtained through interbreeding.

CONCLUSION

CRISPR/Cas9 is a reliable gene-editing tool, and is able to efficiently modify Acvrl1 and create the target mice.

摘要

目的

本研究旨在生成带有 LoxP 序列侧翼的 Acvrl1 基因的突变小鼠，以允许通过 LoxP/Cre 系统对 Acvrl1 进行条件缺失。此类小鼠可能有助于开发脑动静脉畸形 (BAVM) 模型。

方法

使用 CRISPR/Cas9 技术编辑 Acvrl1。设计并构建了两个带有识别位点的单指导 RNA (sgRNA)，位于内含子 3 和 8 上，以及一个与靶基因同源并包含两个 LoxP 序列的供体载体。体外合成的 sgRNA、Cas9 mRNA 和供体载体被注射到小鼠受精卵中，然后将其转移到假孕小鼠中。通过基因分型和测序鉴定新生突变小鼠。