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

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

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。

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 F2generation(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.