Construction of Dmd Gene Mutant Mice and Phenotype Verification in Muscle and Immune Systems
| Autor: | LIANG Min, GUO Yang, WANG Jinjin, ZHU Mengyan, CHI Jun, CHEN Yanjuan, WANG Chengji, YU Zhilan, SHEN Ruling |
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| Jazyk: | čínština |
| Rok vydání: | 2024 |
| Předmět: | |
| Zdroj: | Shiyan dongwu yu bijiao yixue, Vol 44, Iss 1, Pp 42-51 (2024) |
| Druh dokumentu: | article |
| ISSN: | 1674-5817 64576744 |
| DOI: | 10.12300/j.issn.1674-5817.2023.089 |
| Popis: | Objective The aim is to utilize CRISPR/Cas9 gene editing technology to construct Dmd gene mutant mice with a point mutation in exon 23 of the Dmd gene. Subsequently, the phenotypic changes of the mice in muscles and immune systems are analyzed and verified, providing an evaluation model for Duchenne muscular dystrophy and other related diseases. Methods Based on the sequence characteristics of exon 23 of the Dmd gene, small guide RNA (sgRNA) was designed and synthesized. Cas9 mRNA, sgRNA fragments, and oligo donor DNA were microinjected into fertilized eggs of C57BL/6J mice. After transferring the fertilized eggs to surrogate mice, F0 generation mice were born. After mating with F0 generation mice, offspring mice were obtained, and Dmd gene positive mutant (DmdMu/+) mice were obtained after genotype identification. Male hemizygous DmdMu/+(DmdMu/Y) mice were selected for phenotype validation. The body weight of live 3- and 9-month-old mice were recorded. Muscle tension was evaluated through the grid test. Hearts and semitendinosus muscles were collected, and the histopathological changes were observed using HE staining. Further, the expression of Dmd protein in muscle tissue of 9-month-old mice was analyzed by Western blotting. An acute inflammation model was established in DmdMu/Y mice using lipopolysaccharide induction. Peripheral blood from the submandibular vein was collected, and the changes in the proportion of neutrophils and monocytes were detected by flow cytometry. Results The results of genome sequencing and Western blotting confirmed the successful construction of Dmd gene point mutant mice (DmdMu/+ mice). Dmd protein expression was not detected in skeletal muscle and myocardium of DmdMu/+ mice, and it was significantly reduced compared to wild-type C57BL/6J mice (P |
| Databáze: | Directory of Open Access Journals |
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