Combination of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Associated 9 Technique With the PiggyBac Transposon System for Mouse In Utero Electroporation To Study Cortical Development
Combination of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Associated 9 Technique With the PiggyBac Transposon System for Mouse In Utero Electroporation To Study Cortical Development
复制标题
将成簇规则间隔短回文重复序列 (CRISPR) 相关的 9 技术与 PiggyBac 转座子系统相结合，用于小鼠子宫内电穿孔研究皮质发育
DOI：
10.1002/jnr.23776
复制
发表时间：
2016-09-01
影响因子：
4.2
通讯作者：
Zhou, Yan
中科院分区：
文献类型：
--
作者：
Cheng, Man;Jin, Xubin;Zhou, Yan
子宫内电穿孔（IUE）通常用于通过下调或过表达小哺乳动物神经祖细胞（NPC）中感兴趣的基因来研究大脑皮质发育。然而，外源质粒随着时间的推移而丢失或稀释。此外，基于短发夹RNA的基因敲除可能产生导致异常神经元迁移的非特异性效应。通过成簇规则间隔短回文重复序列（CRISPR）/CRISPR-associated 9（Cas9）系统进行基因组工程具有巨大的研究和治疗潜力。在这里，我们将CRISPR/Cas9组件整合到piggyBac（PB）转座子系统（CRISPR/Cas9-PB工具包）中，用于皮质IUE。选择小鼠Sry相关的HMG box-2（Sox 2）基因作为其应用的靶标。大多数转导的皮质NPC早在IUE后3天就耗尽了SOX 2蛋白，而SOX 1和PAX 6的表达保持完整。此外，WT Cas9和D10 A切口酶突变体Cas9 n都显示出相当的敲除效率。用荧光激活细胞分选法纯化转导的皮质细胞，并证实了Sox 2位点的有效基因编辑。因此，CRISPR/Cas9-PB工具包在IUE中的应用是研究皮质NPC及其后代中基因功能的有前途的策略。(C)2016 Wiley Periodicals，Inc.
In utero electroporation (IUE) is commonly used to study cortical development of cerebrum by downregulating or overexpressing genes of interest in neural progenitor cells (NPCs) of small mammals. However, exogenous plasmids are lost or diluted over time. Furthermore, gene knockdown based on short-hairpin RNAs may exert nonspecific effects that lead to aberrant neuronal migration. Genomic engineering by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has great research and therapeutic potentials. Here we integrate the CRISPR/Cas9 components into the piggyBac (PB) transposon system (the CRISPR/Cas9-PB toolkit) for cortical IUEs. The mouse Sry-related HMG box-2 (Sox2) gene was selected as the target for its application. Most transduced cortical NPCs were depleted of SOX2 protein as early as 3 days post-IUE, whereas expressions of SOX1 and PAX6 remained intact. Furthermore, both the WT Cas9 and the D10A nickase mutant Cas9n showed comparable knockout efficiency. Transduced cortical cells were purified with fluorescence-activated cell sorting, and effective gene editing at the Sox2 loci was confirmed. Thus, application of the CRISPR/Cas9-PB toolkit in IUE is a promising strategy to study gene functions in cortical NPCs and their progeny. (C) 2016 Wiley Periodicals, Inc.