Construction of Dmd Gene Mutant Mice and Phenotype Verification in Muscle and Immune Systems

doi:10.12300/j.issn.1674-5817.2023.089

Abstract

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 (Dmd^Mu/+) mice were obtained after genotype identification. Male hemizygous Dmd^Mu/+(Dmd^Mu/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 Dmd^Mu/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 (Dmd^Mu/+ mice). Dmd protein expression was not detected in skeletal muscle and myocardium of Dmd^Mu/+ 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, Dmd^Mu/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 Dmd^Mu/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 Dmd^Mu/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 Dmd^Mu/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 Dmd^Mu/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.

Key words: Dmd gene, CRISPR/Cas9, Duchenne muscular dystrophy, C57BL/6J mice

CLC Number:

Q95-33

Min LIANG, Yang GUO, Jinjin WANG, Mengyan ZHU, Jun CHI, Yanjuan CHEN, Chengji WANG, Zhilan YU, Ruling SHEN. Construction of Dmd Gene Mutant Mice and Phenotype Verification in Muscle and Immune Systems[J]. Laboratory Animal and Comparative Medicine, 2024, 44(1): 42-51.

Figures/Tables 5

Figure 1 Construction strategy of Dmd gene point mutation mice

Table 1 sgRNA and oligo donor DNA as well as PCR primer sequence information

序列名称

Sequence name

序列（5'→3'）

Sequence (5'→3')

小向导RNA

sgRNA

TCTTTGAAAGAGCAACAAAATGG

寡核苷酸供体DNA

Oligo donor DNA

GCTCTGCAAAGTTCTTTGAAAGAGCAATAAAATGGCTTCAACTATCTGAGTGACACTGTGAAGGAGATGGCCAAGAAAGCACCTTCAGAAATATGCCAGAAATATCTGTC

引物I

Primer I

ATGCTTGATATTGAGTAGTTA

引物 II

Primer II

AATAGATTTGGCTTTTGAT

测序引物

Sequencing primer

ATGCTTGATATTGAGTAGTTA

Figure2 Genotype results of Dmd point mutant miceNote：A， identification strategy diagram； B， PCR electrophoresis maps of DmdMu/+ mice； C-D， DNA sequencing results. DmdMu/+， heterozygous point mutant mice； WT， wild-type mice； M， DNA marker.

Figure 3 Phenotypic validation of DmdMu/Y miceNote：A-B, The expression levels of Dmd protein in semitendinosus (ST) and heart of DmdMu/Y mice were detected by Western blotting [wild-type C57BL/6J mice (WT) were used as the control group; GAPDH was internal control protein; n=3, *P<0.05, **P<0.01]; C, the body weight of 3-month and 9-month old DmdMu/Y mice [wild-type C57BL/6J mice (WT) were used as the control group; n=9, **P<0.01]; D, HE staining results of muscle tissue pathologic changes in 3-month and 9-month old DmdMu/Y mice [wild-type C57BL/6J mice (WT) were used as the control group; the gap between skeletal (ST) and myocardial (Heart) muscles of 9-month old DmdMu/Y mice became wider and the size of muscles varied; Infiltrating inflammatory cells were observed (sigh by arrow);n=3，bar=100 μm]. E, Schematic diagram of grid test; F, The statistical chart of the grid test [3-month old, DmdMu/Y mice n=6, WT mice n=5; 9-month old, DmdMu/Y mice n=8, WT mice n=5, *P<0.05, **P<0.01].

Figure 4 The proportion of neutrophils and monocytes in the blood of DmdMu/Y mice of LPS-induced acute inflammation model detected by flow cytometryNote： A-B, Changes in neutrophils ratio (A) and monocyte ratio (B) in 3-month old DmdMu/Y mice after injection of lipopolysaccharide (LPS) for 0 and 72 hours; C-D, Changes in neutrophils ratio (C) and monocyte ratio (D) in 9-month old DmdMu/Y mice after injection of LPS for 0 and 72 hours. The wild-type C57BL/6J mice (WT) were used as the control group; n=5, *P<0.05, **P<0.01.

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