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

doi:10.13560/j.cnki.biotech.bull.1985.2021-0647

Abstract

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

YAN Jiong, FENG Chen-yi, GAO Xue-kun, XU Xiang, YANG Jia-min, CHEN Zhao-yang. Construction of Homozygous Plin1-knockout Mouse Model and Phenotype Analysis Based on CRISPR/Cas9 Technology[J]. Biotechnology Bulletin, 2022, 38(3): 173-180.

Figures/Tables 10

Fig. 1 Schematic diagram of sgRNA knocking out exon 2 of Plin1 gene The Plin1 gene transcript is NM_001113471.1

Table 1 sgRNA sequence

名称 Name	序列 Sequence（5'-3'）	PAM序列 PAM sequence
sgRNA-Plin1-1	TTGGAGGGCGAATGTTACAT	AGG
sgRNA-Plin1-2	GAACTTCAGGCGTATGACCC	TGG

Fig. 2 Strategies for genotype identification of Plin1 knoc-kout mice The Plin1 gene transcript is NM_001113471.1

Table 2 Primer sequence for genotyping

名称Name	序列Sequence（5'-3'）	大小Size/bp
Plin1-F	CTGAGAGAAGGCTTAACCTTGCTGG	25
Plin1-R₁	AGCTTTCCATCCTGCAAGTGAGTCAG	26
Plin1-R₂	AGGTGGCAAGGACAGAGACAGTGAG	25

Fig. 3 Construction and identification of sgRNA expression vector A：Digestion results of sgRNA expression vector（Lane 2 and 4：sgRNA-Plin1-1,2 vectors. Lane 1 and 3：Enzyme fragment. M：DNA marker）. B：Sequencing results of sgRNA expression vector. C：In vitro transcriptional fragment electrophoresis results of sgRNA（Lane 1-3：sgRNA-Plin1-1 transcription products. Lane 4-6 lane：sgRNA-Plin1-2 transcription products. M：DNA marker）

Fig. 4 Genotype identification of F0 and F1 generation mice A：Electrophoretic results of Plin1-F1/R1 amplified from F0 generation positive mice;lane 1-12：F0 positive mice;WT：wild type control;M. DNA marker. B-C：Electrophoretic results of Plin1-F1/R1 amplified from F1 generation mice;lane 1-13：F1 generation mice;WT：wild type control. M：DNA marker. D：gene sequencing results of F0 and F1 generation mice

Fig. 5 Genotype identification of F2 generation mice A：Electrophoretic results of Plin1 gene amplified by Plin1-F1/R1. B：Electrophoretic results of Plin1 gene amplified by Plin1-F1/R2. Lane 1-17：Plin1 gene fragment of F2 generation mice. White arrow：Homozygous. Black arrow：Wild type

Table 3 Physical parameters of mice in each group

组别/指标 Group/index	体重 Body weight/g	体长 Body length/cm	摄食量 Food intake/g
野生型WT	22.95±0.55	8.88±2.17	16.26±2.13
杂合型Plin1^+/-	23.20±1.57	9.31±3.49	15.43±3.03
纯合型Plin1^-/-	18.34±0.35*	8.72±1.34	15.67±2.59

Fig. 6 Relative expressions of PLIN1 mRNA in the various tissues of mice A：Relative level of PLINI mRNA in liver. B：Relative level of PLINI mRNA in skeletal muscle. C：Relative level of PLINI mRNA in white adipose tissue. **：Com-pared with Plin1+/-. P < 0.05. *：Compared with WT,P < 0.05. The same below

Fig.7 Protein expressions of PLIN1 in the various tissues of mice

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