实验动物与比较医学2024，Vol.44Issue(1)：42-51,10.DOI:10.12300/j.issn.1674-5817.2023.089
Dmd基因突变小鼠构建及在肌肉及免疫系统的表型验证
Construction of Dmd Gene Mutant Mice and Phenotype Verification in Muscle and Immune Systems
摘要
Abstract
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＜0.05).Compared with wild-type mice of the same background,DmdMu/Y mice at 3 and 9 months of age showed significant weight loss(P＜0.01)and decreased muscle tension(P＜0.05).9-month-old DmdMu/Y mice exhibited significant pathological changes in skeletal muscle and myocardium,including widening of intermuscular space.Under normal condition,compared with wild-type mice,the proportion of neutrophils and monocytes in the peripheral blood of 3-month-old DmdMu/Y mice was significantly lower than that of wild-type mice(P＜0.01).After lipopolysaccharide stimulation,the proportion of neutrophils in peripheral blood of 3-month-old DmdMu/Y mice remained significantly lower compared to that of wild-type mice(P＜0.01).The proportion of neutrophils in peripheral blood of 9-month-old DmdMu/Y mice significantly decreased after lipopolysaccharide induction(P＜0.01),with a trend of change observed in monocytes between groups.Conclusion The successful construction of the Dmd gene mutant mouse model has confirmed the vital function of Dmd gene in maintaining normal muscle tissue morphology and muscle tone.It preliminarily indicated that Dmd gene deletion could significantly reduce the proportion of neutrophils in peripheral blood,offering a new perspective for the study of immune system alterations in Duchenne muscular dystrophy patients.关键词
Dmd基因/CRISPR/Cas9/杜氏肌营养不良症/C57BL/6J小鼠Key words
Dmdgene/CRISPR/Cas9/Duchenne muscular dystrophy/C57BL/6J mice分类
生物科学引用本文复制引用
梁敏,郭洋,王津津,朱梦妍,池骏,陈艳娟,王成稷,喻智澜,沈如凌..Dmd基因突变小鼠构建及在肌肉及免疫系统的表型验证[J].实验动物与比较医学,2024,44(1):42-51,10.基金项目
上海市科技创新行动计划-实验动物专项课题"人源化小鼠模型的建立和评价平台建设"(22140900102) （22140900102）
