Construction, breeding, and gene identification of TREM2 knockout mice
10.19405/j.cnki.issn1000-1492.2025.06.001
- Author:
Rong Huang
1
;
Xinxin Zhao
1
;
Hui Xue
2
;
Mengjuan Zhu
2
;
Jiajie Tu
2
;
Xinming Wang
1
Author Information
1. School of Pharmacy , Anhui Medical University , Hefei 230032 ; Dept of Pharmacy , The First Afiliated Hospital of Anhui Medical University , Hefei 230022
2. Institute of Clinical Pharmacology , Anhui Medical University , Hefei 230032
- Publication Type:Journal Article
- Keywords:
triggering receptor expressed on myeloid cells 2;
CRISPR / Cas9;
gene knockout;
polymerase chain reaction;
agarose gel electrophoresis;
genotype identification
- From:
Acta Universitatis Medicinalis Anhui
2025;60(6):977-983
- CountryChina
- Language:Chinese
-
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. Polymerase 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-/- mice was reduced(P-/- mice.
Conclusion:TREM2-/- mice are successfully constructed and bred, a reliable genotype identification method is established, the genetic stability of the mouse model is verified, which will provide an important genetic animal model for the study of TREM2 gene function.
- Full text:202602091039038143TREM2基因敲除小鼠的构建、繁育和基因鉴定_黄蓉.pdf
