基于CRISPR/Cas9技术构建Plin1基因敲除小鼠模型及表型分析

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

摘要/Abstract

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

燕炯, 冯晨毅, 高学坤, 许祥, 杨佳敏, 陈朝阳. 基于CRISPR/Cas9技术构建Plin1基因敲除小鼠模型及表型分析[J]. 生物技术通报, 2022, 38(3): 173-180.

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.

图/表 10

图1 sgRNA敲除Plin1基因2号外显子的示意图 Plin1基因转录本为NM_001113471.1

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

表1 sgRNA序列

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

图2 Plin1基因小鼠敲除基因型鉴定的策略 Plin1基因转录本为NM_001113471.1。

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

表2 基因型鉴定的引物序列

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

图3 sgRNA表达载体构建与鉴定 A：sgRNA表达载体的酶切结果（2,4泳道：sgRNA-Plin1-1、2载体;1,3泳道：酶切片段;M：DNA marker）;B：sgRNA表达载体测序结果;C：sgRNA体外转录片段电泳结果（1-3泳道：sgRNA-Plin1-1转录产物;4-6：sgRNA-Plin1-2转录产物;M：DNA marker）

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）

图4 F0和F1代小鼠基因型鉴定 A：F0代阳性小鼠Plin1-F1/R1扩增Plin1基因的电泳结果;1-12泳道：F0阳性小鼠;WT：野生型对照;M：DNA marker;B-C：F1代小鼠Plin1-F1/R1扩增Plin1基因的电泳结果。1-13泳道：F1代小鼠;WT：野生型对照;M：DNA marker;D：F0、F1代小鼠基因测序结果

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

图5 F2代小鼠基因型鉴定 A：Plin1-F1/R1扩增Plin1基因的电泳结果;B：Plin1-F1/R2扩增Plin1基因的电泳结果;1-17泳道：F2代小鼠Plin1基因片段;白色箭头：纯合型;黑色箭头：野生型

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

表3 各组小鼠体格参数

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

图6 小鼠各组织中PLIN1 mRNA的相对表达量 A：肝脏中PLIN1 mRNA相对表达量;B：骨骼肌中PLIN1 mRNA相对表达量;C：白色脂肪组织中PLIN1 mRNA相对表达量;**：相比于Plin1+/-,P < 0.05;*：相比于WT,P < 0.05,下同

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

图7 小鼠各组织中PLIN1的蛋白表达水平

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

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