基于CRISPR/Cas9构建Msx1-Cre小鼠模型

›› 2020, Vol. 11 ›› Issue (3): 156-160.

基于CRISPR/Cas9构建Msx1-Cre小鼠模型

陈毅斌,李琳君,董碧莹,林陈胜

福建省发育与神经生物学重点实验室，福建师范大学生命科学学院

收稿日期:2020-07-19 修回日期:2020-08-22 出版日期:2020-09-25 发布日期:2020-09-30
通讯作者: 林陈胜 E-mail:linchensheng@fjnu.edu.cn
基金资助:
福建省自然科学基金面上项目

Construction of Msx1Cre mouse model based on CRISPR/Cas9

Received:2020-07-19 Revised:2020-08-22 Online:2020-09-25 Published:2020-09-30

摘要/Abstract

摘要： 目的：构建肌节同源盒基因1（Msx1）-Cre小鼠模型。方法：基于CRISPR/Cas9技术，设计靶向切割Msx1基因启动子下游的单链向导RNA（sgRNA），并构建包含Cre序列的同源重组片段；构建sgRNA载体质粒，转染至小鼠胚胎成纤维细胞（NIH3T3），并在细胞水平验证sgRNA的打靶效率和脱靶情况；将靶向切割Msx1基因的sgRNA、Cre同源重组片段及Cas9蛋白等混合溶液经体外显微注射至C57BL/6J小鼠的受精卵，3周后获得小鼠，利用PCR技术鉴定获得的F0代Msx1-Cre小鼠，并将构建成功的Msx1-Cre模型小鼠与Rosa26R-mTmG模型小鼠交配，进行功能验证。结果：设计的sgRNA能够有效定点切割Msx1基因启动子下游靶位点；成功获得Msx1-Cre小鼠模型；Msx1-Cre模型小鼠与Rosa26R-mTmG模型小鼠交配，子代小鼠免疫荧光结果显示，小鼠绿色荧光蛋白（GFP）特异定位于表达MSX1细胞的细胞膜上。结论：成功构建了Msx1-Cre小鼠模型，可用于示踪Msx1基因以及在表达MSX1的牙间充质细胞中进行条件性敲除或过表达实验来研究牙发育过程中特定基因的功能

关键词: CRISPR/Cas9, Msx1, Cre, 小鼠模型

Abstract: Objective：To construct the Msx1-Cre mouse model. Methods：Design two sgRNA in the CRISPR/Cas9 system that target the Msx1 gene and construct homologous recombination fragments containing Cre sequences; Construct sgRNA vector plasmids and verify the target efficiency and off-target situation of sgRNA at the cellular level; Obtain F0 generation mice knocked into Cre sequence of Msx1 gene by microinjection method; Identification and functional verification of mouse models. Results：The designed sgRNA effectively cut the target site downstream of the Msx1 promoter in a targeted manner; We successfully obtained the Msx1Cre mouse model by microinjection; GFP was specifically located on the membrane of Msx1+ cells in Msx1Cre;R26RmTmG mice. Conclusions：The Msx1Cre mouse model was successfully constructed based on the CRISPR/Cas9 system.

Key words: CRISPR/Cas9, Msx1, Cre, mouse models

陈毅斌李琳君董碧莹林陈胜. 基于CRISPR/Cas9构建Msx1-Cre小鼠模型[J]. 口腔生物医学, 2020, 11(3): 156-160.

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