生物技术通报 ›› 2022, Vol. 38 ›› Issue (3): 173-180.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0647

• 研究报告 • 上一篇    下一篇

基于CRISPR/Cas9技术构建Plin1基因敲除小鼠模型及表型分析

燕炯1(), 冯晨毅1, 高学坤1, 许祥1, 杨佳敏1, 陈朝阳2()   

  1. 1.山西医科大学公共卫生学院营养与食品卫生学教研室,太原 030001
    2.山西医科大学实验动物中心,太原 030001
  • 收稿日期:2021-05-19 出版日期:2022-03-26 发布日期:2022-04-06
  • 作者简介:燕炯,男,博士,副教授,研究方向:营养相关疾病分子学;E-mail: yanjiong@126.com
  • 基金资助:
    国家自然科学基金项目(31772551);山西省自然科学基金(201901D111182)

Construction of Homozygous Plin1-knockout Mouse Model and Phenotype Analysis Based on CRISPR/Cas9 Technology

YAN Jiong1(), FENG Chen-yi1, GAO Xue-kun1, XU Xiang1, YANG Jia-min1, CHEN Zhao-yang2()   

  1. 1. Department of Nutrition and Food Hygiene School of Public Health,Shanxi Medical University,Taiyuan 030001
    2. Laboratory Animal Center,Shanxi Medical University,Taiyuan 030001
  • Received:2021-05-19 Published:2022-03-26 Online:2022-04-06

摘要:

利用CRISPR/Cas9技术构建纯合围脂滴蛋白(Plin1)基因敲除小鼠模型,并初步分析其表型。针对Plin1基因2号外显子前后序列设计sgRNA并构建表达载体,体外转录获得sgRNA后与Cas9蛋白混合,显微注射至小鼠受精卵中并进行胚胎移植。出生小鼠经测序及PCR基因型鉴定获得F0代阳性小鼠;令F0代小鼠与野生型小鼠杂交,获得F1代杂合子小鼠;通过F1代杂合小鼠近交,获得F2代纯合子小鼠模型。常规饲喂同窝别Plin1基因敲除纯合小鼠、杂合小鼠和野生型小鼠,并测量小鼠体重、身长等体格参数;定量实时聚合酶链反应(Q-RT-PCR)和蛋白质印迹(WB)分别检测每个组织中Plin1基因在mRNA和蛋白质水平的表达。敲除了小鼠Plin1基因741 bp的片段(包含了2号外显子在内);Plin1基因敲除小鼠PLIN1 mRNA和蛋白表达水平均显著降低(P < 0.05);常规喂养4周后,相比于野生型及杂合型小鼠,纯合型Plin1基因敲除小鼠体重显著降低(P < 0.05)。成功构建了Plin1基因敲除小鼠模型;初步表型分析发现,Plin1基因敲除小鼠与野生型小鼠相比较为瘦弱。

关键词: 围脂滴蛋白, CRISPR/Cas9技术, 基因敲除, 脂质代谢

Abstract:

The objectives are to construct a homozygous Plin1-knockout mouse model by CRISPR/Cas9 technology and to preliminarily analyze their phenotypic changes. A pair of sgRNA based on the sequence before and after exon 2 of Plin1 gene was designed and its corresponding plasmid expression vectors were constructed. The sgRNA,obtained by in vitro transcription of the expression vector,mixed with Cas9 protein was delivered to the mouse fertilized egg by microinjection technology,and then the embryo transfer of the fallopian tube was conducted. F0 generation positive mice were obtained by gene sequencing and PCR genotype identification and screening after the first generation of mice was born. Then F0 generation mice were crossed with wild-type mice to obtain F1 generation heterozygous mice. The F1 generation of heterozygous mice was inbred between male and female,and the F2 generation homozygous mouse model was obtained. Routinely feeding homozygous Plin1-knockout mice,heterozygous and wild-type mice in the same litter,their physical parameters such as body weight and length of the mice were measured. Quantitative Real-time-Polymerase Chain Reaction and Western blot were to detect the expressions of Plin1 at mRNA and protein levels in each tissue respectively. As results,741bp(including exon 2)fragment of Plin1 gene in the mice was knocked out. Compared with wild-type mice,the PLIN1 mRNA and protein expressions of the Plin1-knockout mice significantly reduced(P <0.05). After 4 weeks of routine feeding,compared with wild-type and heterozygous mice,the weight of homozygous Plin1-knockout mice significantly reduced(P < 0.05). In conclusion,a mouse model of Plin1-knockout mice is successfully constructed,and preliminary phenotypic analysis found that Plin1-knockout mice were thinner than wild-type ones.

Key words: PLIN1, CRISPR/Cas9, gene knockout, lipid metabolism