Construction and phenotypic study of heterozygous knockout mice of tumor necrosis factor receptor associated factor 2

Acta Universitatis Medicinalis Anhui     font:big middle small

Found programs: National Natural Science Foundation of China (Nos . 82104187 , 82173824)

Authors:Wang Weikang , Zuo Shujun , Gu Jintao , Guo Fuyuan , Guo Haozhou , Han Chenchen , Wei Wei

Keywords:TRAF2 ; CRISPR/Cas9 ; gene knockout; genetically engineered mice; genotyping; phenotypic study

DOI:10.19405/j.cnki.issn1000-1492.2025.07.018

〔Abstract〕 To generate heterozygous TRAF2 knockout mice , the CRISPR/Cas9 technology was suc- cessfully employed . These mice were served as a valuable model to explore the pathological mechanisms underlying inflammatory and immune disorders mediated by abnormal TNF-α-TRAF2 signaling and to develop new therapeutic targets . Methods A vector targeting the knockout of the TRAF2 gene was constructed . Lead RNA and Cas9 Mrna were introduced into the fertilized eggs of C57BL/6JGpt mice through microinjection to mediate the TRAF2 gene mutation in mice . The mouse tail protein was extracted and the genotype of the F0 generation was determined by PCR and Western blot. TRAF2 + / - mice were successfully obtained . F0 generation mice were backcrossed with C57BL/6JGpt wild-type mice to obtain stable TRAF2 + / - mice for propagation and subsequent experiments . The body weight of TRAF2 + / - mice was detected; Western blot was used to detect the expression of TRAF2 in the spleen , liver and kidney tissues of TRAF2 + / - mice . The development of spleen , liver and kidney tissues in TRAF2 + / - mice was detected by HE staining. Results PCR identification using specific primers demonstrated that TRAF2 + / - mice exhibited a target band at 679 bp . Western blot analysis results indicated that , compared with the WT group , the expression of TRAF2 in the tail protein of TRAF2 + / - mice was significantly reduced( P < 0. 01) . TRAF2 + / - mice had a lower body weight compared to their littermate WT mice(P < 0. 05) . Western blot analysis showed that , relative to the WT group , the expression of TRAF2 protein in the spleen , liver , and kidney tissues of TRAF2 + / - mice was decreased(P < 0. 01) . HE staining results indicated that there were no significant differences in cellular morphology in the spleen , liver , and kidney tissues between TRAF2 + / - mice and WT mice . Conclusion The successful construction of TRAF2 + / - mice has provided an important animal model for exploring the role of TRAF2 in developmental regulation , revealing the mechanism of inflammatory immune diseases mediated by abnor- mal TNF-α-TRAF2 signaling , and screening related drug targets .