安徽医科大学学报2025，Vol.60Issue(6)：977-983,7.DOI:10.19405/j.cnki.issn1000-1492.2025.06.001
TREM2基因敲除小鼠的构建、繁育和基因鉴定
Construction,breeding,and gene identification of TREM2 knockout mice
摘要
Abstract
Objective To construct triggering receptor expressed on myeloid cells 2(TREM2)gene knockout(TREM2-/-)mice using CRISPR/Cas9 technology,to breed TREM2-/-mice and to analyze the genotype of TREM2-/-mice.Methods CRISPR/Cas9 technology was used to selectively knock out exon 2-3 regions of TREM2 gene to construct a TREM2-/-mouse model,and the genetic background of all mice was C57BL/6J.Poly-merase chain reaction(PCR)was used to identify the genotype of mice.Quantitative real-time PCR(qPCR)and Western blot were used to detect the expression level of TREM2 in major tissues of mice,and the authenticity and scientific nature of PCR identification results were verified from mRNA level and protein level.According to the sgRNA sequence,the possible off-target sites were predicted on the CCTop website,and the tail DNA of mice was extracted and amplified by PCR and then Sanger sequencing was performed to detect whether there was off-target effect in TREM2-/-mice.Results TREM2-/-mice were successfully constructed by CRISPR/Cas9 technology,and the mice were genotyped.The results of agarose gel electrophoresis showed that the mouse genotype with only 415 bp band amplified was wild type(WT),the mouse genotype of the 449 bp band amplified only was TREM2-/-,and the mouse genotypes amplified with 415 bp and 449 bp double bands were heterozygous.qPCR results showed that compared with WT mice,the mRNA expression of TREM2 in heart and brain tissues of TREM2-/-mice was down-regulated(P＜0.01),and the mRNA expression of TREM2 in thymus and lung tissues also showed a downward trend(P＜0.05).Western blot results showed that compared with WT mice,the protein expression level of TREM2 in heart,brain,thymus,lung tissues and peripheral blood mononuclear cell(PBMC)of TREM2-/-mice was reduced(P＜0.01).Sanger sequencing results showed no off-target effects in TREM2-/-mice.Conclusion TREM2-/-mice are successfully constructed and bred,a reliable genotype identification meth-od is established,the genetic stability of the mouse model is verified,which will provide an important genetic ani-mal model for the study of TREM2 gene function.关键词
髓细胞触发受体2/CRISPR/Cas9/基因敲除/聚合酶链式反应/琼脂糖凝胶电泳/基因型鉴定Key words
triggering receptor expressed on myeloid cells 2/CRISPR/Cas9/gene knockout/polymerase chain reaction/agarose gel electrophoresis/genotype identification分类
医药卫生引用本文复制引用
黄蓉,赵鑫鑫,薛慧,朱梦娟,涂佳杰,王鑫铭..TREM2基因敲除小鼠的构建、繁育和基因鉴定[J].安徽医科大学学报,2025,60(6):977-983,7.基金项目
国家自然科学基金(编号:82104185) （编号:82104185）
安徽省高校科研项目(编号:2022AH051153) （编号:2022AH051153）
安徽省自然科学基金(编号:2008085QH400) （编号:2008085QH400）
抗炎免疫药物教育部重点实验室(安徽医科大学)开放课题(编号:KFJJ-2020-03) （安徽医科大学）
安徽医科大学校科研基金(编号:2021xkj148) National Natural Science Foundation of China(No.82104185) （编号:2021xkj148）
Natural Science Research Pro-ject of Anhui Educational Committee(No.2022AH051153) （No.2022AH051153）
Natural Science Foundation of Anhui Province(No.2008085QH400) （No.2008085QH400）
Open Project of Key Laboratory of Anti-inflammatory and Immune Medicine,Ministry of Educa-tion,at Anhui Medical University(No.KFJJ-2020-03) （No.KFJJ-2020-03）
Scientific Research Project of Anhui Medical University(No.2021xkj148) （No.2021xkj148）
