Found programs: National Natural Science Foundation of China (No . 82104185) ; Natural Science Research Pro- ject of Anhui Educational Committee (No . 2022AH051153) ; Natural Science Foundation of Anhui Province( 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) ; Scientific Research Project of Anhui Medical University (No . 2021xkj148)
Authors：Huang Rong1 , 2 , Zhao Xinxin1 , 2 , Xue Hui3 , Zhu Mengjuan3 , Tu Jiajie3 , Wang Xinming1 , 2
Keywords：triggering receptor expressed on myeloid cells 2 ; CRISPR / Cas9 ; gene knockout; polymerase chain reaction; agarose gel electrophoresis; genotype identification
DOI：10.19405/j.cnki.issn1000-1492.2025.06.001
〔Abstract〕 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 .