Department of Anesthesiology, Huashan Hospital, Fudan University, Shanghai 200040, China.
Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai 200040, China.
Mol Cell Probes. 2018 Feb;37:32-38. doi: 10.1016/j.mcp.2017.11.003. Epub 2017 Nov 10.
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.
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.
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.
CRISPR/Cas9 is a reliable gene-editing tool, and is able to efficiently modify Acvrl1 and create the target mice.
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。
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。
以 8.7%的成功率生成了 2 只带有 floxed Acvrl1 等位基因的小鼠。通过杂交获得了健康且有生育能力的靶标小鼠。
CRISPR/Cas9 是一种可靠的基因编辑工具，能够有效地修饰 Acvrl1 并创建靶标小鼠。
