Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (3): 173-180.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0647

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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 Online:2022-03-26 Published:2022-04-06
  • Contact: CHEN Zhao-yang E-mail:yanjiong@126.com;ccytycn@163.com

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