吉林大学学报(医学版) ›› 2022, Vol. 48 ›› Issue (2): 271-276.doi: 10.13481/j.1671-587X.20220201

• 基础研究 •    下一篇

基于CRISPR/Cas9技术构建LDLR基因敲除的免疫缺陷小鼠模型及其表型分析

王兆卫,吕亚楠,胡正,杨永广()   

  1. 吉林大学第一医院 器官再造与移植教育部重点实验室,吉林 长春 130061
  • 收稿日期:2021-08-30 出版日期:2022-03-28 发布日期:2022-05-10
  • 通讯作者: 杨永广 E-mail:yongg@jlu.edu.cn
  • 作者简介:王兆卫(1995-),男,河南省项城市人,在读硕士研究生,主要从事人源化小鼠模型方面的研究。
  • 基金资助:
    国家自然科学基金专项项目(81941008)

Construction of LDLR gene knockout immunodeficient mouse model and its phenotypic analysis based on CRISPR/cas9 technology

Zhaowei WANG,Yanan LYU,Zheng HU,Yongguang YANG()   

  1. Key Laboratory of Organ Regeneration & Transplantation of Ministry of Education,First Hospital,Jilin University,Changchun 130061,China
  • Received:2021-08-30 Online:2022-03-28 Published:2022-05-10
  • Contact: Yongguang YANG E-mail:yongg@jlu.edu.cn

摘要: 目的

基于CRISPR/Cas9技术构建低密度脂蛋白受体(LDLR)基因敲除的免疫缺陷小鼠模型,并对血液胆固醇水平进行分析评价,为构建具有高脂血症的免疫系统人源化小鼠模型提供新方法。

方法

基于CRISPR/Cas9技术,将有效识别LDLR基因外显子2和18的sgRNA/Cas9 mRNA注射到NOD SCID小鼠的受精卵中,通过对新生小鼠基因型鉴定筛选得到基因敲除的F0代阳性(LDLR+/-,Aa)小鼠,再将此小鼠与NOD SCID(LDLR+/+,AA)小鼠繁育,鉴定得到能稳定遗传基因型的F1代(Aa)小鼠。将F1代阳性杂合小鼠与NOD SCID小鼠繁育,获得大量基因序列完全相同的F2代(Aa)小鼠,在F2代间进行大规模繁育,获得的F3代小鼠依据基因型和性别进行分组,分别为雄性AA、雄性Aa、雌性AA和雌性Aa,对体质量进行监测,同时采集外周血进行血液胆固醇水平检测。

结果

通过上述构建方法获得了NOD SCID LDLR+/-(Aa)小鼠,经过8周的体质量检测,Aa杂合子基因型在生长发育过程中并不影响小鼠体质量,雌性Aa的胆固醇水平为(100.80±4.42)mg·dL-1,雄性Aa的胆固醇水平为(120.56±11.16)mg·dL-1,与阴性对照(基因型为AA的小鼠)比较,胆固醇水平明显升高(P<0.05);雌性AA的胆固醇水平为(60.78±2.11)mg·dL-1,雄性AA的胆固醇水平为(75.43±10.06)mg·dL-1,两者比较差异有统计学意义(P<0.05)。

结论

在不影响小鼠体质量的前提下,通过CRISPR/Cas9技术在NOD SCID 背景下成功构建出了胆固醇水平自发升高的小鼠模型。

关键词: 高脂血症, 免疫系统人源化小鼠, 低密度脂蛋白受体, 胆固醇, CRISPR/Cas9技术

Abstract: Objective

To construct an immundeficient mouse model with low density lipoprotein receptor (LDLR) gene knockout by CRISPR/cas9 technology and analyze and evaluate the blood cholesterol level,and to provide a new method for constructing a humanized mouse model of immune system with hyperlipidemia.

Methods

Using CRISPR/cas9 technology, sgRNA/cas9 mRNA effectively identifying exons 2 and 18 of LDLR gene was injected into the fertilized eggs of NOD SCID mice. The gene knockout F0 positive (LDLR+/+, AA) mice were screened by genotyping of neonatal mice, and the mice were bred with NOD SCID (LDLR+/+, AA) mice to identify the F1 generation (AA) with stable genotypes mice. The F1 positive heterozygous mice and NOD SCID mice were bred to obtain a large number of F2 generation (AA) mice with exactly the same gene sequence. Large scale breeding was carried out between F2 generations.The F3 generation mice were grouped according to genotype and gender, respectively: male AA, male Aa, female AA and female Aa.The body weights were monitored, and the peripheral blood was collected for blood cholesterol level detection.

Results

The NOD SCID LDLR+/- (AA) mice were obtained by the above construction method. After 8 weeks of weight detection, it was found that Aa heterozygous genotype did not affect the body weight of mice during growth and development process. The cholesterol level of female Aa was (100.80±4.42) mg·dL-1 and male Aa was (120.56±11.16) mg·dL-1, compared with negative control (AA mice),the cholesterol levels were significantly increased(P<0.05);the cholesterol levels of female AA and male AA were(60.78±2.11) and (75.43±10.06) mg·dL-1,and the difference between them was statistically significant (P<0.05).

Conclusion

Without affecting the body weight of mice, a mouse model with spontaneous increase of cholesterol level is successfully constructed by using CRISPR/cas9 technology under the background of NOD SCID, which can be used as the basis for humanization.

Key words: Hyperlipidaemia, Immune system humanized mice, Low density lipoprotein receptor, Cholesterol, CRISPR/cas9

中图分类号: 

  • Q78