Dmd基因突变小鼠构建及在肌肉及免疫系统的表型验证

doi:10.12300/j.issn.1674-5817.2023.089

实验动物与比较医学 ›› 2024, Vol. 44 ›› Issue (1): 42-51.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

梁敏¹(), 郭洋¹, 王津津², 朱梦妍¹, 池骏², 陈艳娟¹, 王成稷¹, 喻智澜¹, 沈如凌¹()()

^1.上海实验动物研究中心, 上海 201203
^2.上海南方模式生物科技股份有限公司, 上海 201318

收稿日期:2023-06-27 修回日期:2023-11-28 出版日期:2024-02-25 发布日期:2024-03-07
通讯作者: 沈如凌（1981—），女，博士，副研究员，研究方向：模式动物模型研发及表型研究（肿瘤免疫和神经免疫方向）。E-mail： shenruling@slarc.org.cn。ORCID： 0000-0002-7529-810X
作者简介:梁敏（1981—），男，博士，助理研究员，研究方向：模式动物模型研发及表型研究（神经免疫方向）。E-mail： liangmin@slarc.org.cn
基金资助:
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)

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

Min LIANG¹(), Yang GUO¹, Jinjin WANG², Mengyan ZHU¹, Jun CHI², Yanjuan CHEN¹, Chengji WANG¹, Zhilan YU¹, Ruling SHEN¹()()

^1.Shanghai Laboratory Animal Research Center, Shanghai 201203, China
^2.Shanghai Model Organisms Inc. Shanghai 201318, China

Received:2023-06-27 Revised:2023-11-28 Published:2024-02-25 Online:2024-03-07
Contact: SHEN Ruling (ORCID: 0000-0002-7529-810X), E-mail: shenruling@slarc.org.cn

摘要/Abstract

摘要：

目的利用CRISPR/Cas9基因编辑技术构建抗肌萎缩蛋白（dystrophin，Dmd）基因第23号外显子点突变的Dmd基因突变小鼠，分析并验证该小鼠在肌肉及免疫系统中的表型变化，为杜氏肌营养不良症等疾病提供评价模型。方法针对Dmd基因23号外显子序列特征，设计合成小向导RNA（small guide RNA，sgRNA）；将Cas9 mRNA、sgRNA片段和oligo donor DNA显微注射到C57BL/6J小鼠受精卵中，并将该受精卵移植至代孕鼠后出生获得F0代小鼠；F0代小鼠通过交配获得子代小鼠后，基因型鉴定筛选得到Dmd基因阳性突变小鼠（命名为Dmd^Mu/+小鼠）。选择3月龄和9月龄的雄性半合子Dmd^Mu/+小鼠（命名为Dmd^Mu/Y小鼠）和野生型C57BL/6J小鼠（作为对照）用于表型验证：称量记录活体小鼠体重，并通过悬挂实验检测小鼠肌张力；采集心脏和半腱肌，采用HE染色法观察组织病理学变化，进一步通过蛋白质印迹法检测9月龄小鼠肌肉组织内Dmd蛋白的表达情况。利用脂多糖诱导建立Dmd^Mu/Y小鼠急性炎症模型，采集小鼠颌下静脉血，采用流式细胞术检测外周血中性粒细胞和单核细胞比例变化。结果基因组测序和蛋白质印迹结果表明Dmd基因点突变小鼠（即Dmd^Mu/+小鼠）构建成功，Dmd^Mu/+小鼠骨骼肌和心肌中未见Dmd蛋白表达，与野生型C57BL/6J小鼠相比明显下降（P<0.05）。与背景相同的野生型小鼠相比较，3月龄和9月龄的雄性半合子突变小鼠（Dmd^Mu/Y小鼠）体重明显减轻（P<0.01），肌张力显著下降（P<0.05）；9月龄Dmd^Mu/Y小鼠的骨骼肌和心肌发生肌间隙变宽等明显病变。未注射脂多糖时，与野生型小鼠相比，3月龄Dmd^Mu/Y小鼠的外周血中性粒细胞和单核细胞比例显著降低（P<0.01）；经脂多糖诱导后，3月龄Dmd^Mu/Y小鼠的外周血中性粒细胞比例仍然较野生型小鼠显著降低（P<0.01），而9月龄Dmd^Mu/Y小鼠的外周血中性粒细胞比例在脂多糖诱导后显著升高（P<0.05），但较野生型小鼠仍显著降低（P<0.01）；同月龄Dmd^Mu/Y小鼠经脂多糖诱导后外周血单核细胞比例与野生型小鼠相比仅有偏低趋势（P>0.05）。结论成功构建了Dmd基因突变小鼠模型，证实该基因具有维系肌肉组织正常形态和肌张力等重要功能，并初步发现该基因缺失会减少外周血液中性粒细胞比例，为后续研究杜氏肌营养不良症患者的免疫系统异变机制提供新的思路。

关键词: Dmd基因, CRISPR/Cas9, 杜氏肌营养不良症, C57BL/6J小鼠

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

中图分类号:

Q95-33

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.

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.

图/表 5

图1 Dmd基因点突变小鼠的构建策略示意图

Figure 1 Construction strategy of Dmd gene point mutation mice

表1 sgRNA、oligo donor DNA和PCR引物序列信息

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

图2 Dmd基因点突变小鼠基因型鉴定注：A，鉴定策略示意图；B，点突变小鼠PCR产物电泳结果；C～D，DNA测序结果（DmdMu/+ 为杂合点突变小鼠，WT为野生型C57BL/6J小鼠，M为DNA分子量标准物）

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.

图3 DmdMu/Y小鼠表型验证注：A～B，蛋白质印迹法检测DmdMu/Y小鼠半腱肌（ST）和心脏组织中Dmd蛋白水平［野生型C57BL/6J小鼠（WT）作为对照，GAPDH为内参蛋白，n=3， *P<0.05， **P<0.01］；C，3月龄和9月龄DmdMu/Y小鼠和野生型C57BL/6J小鼠（WT）体重测量结果［野生型C57BL/6J小鼠（WT）作为对照，n=9， **P<0.01］；D，HE染色检测3月龄和9月龄DmdMu/Y小鼠的肌肉病理学变化［与野生型C57BL/6J小鼠（WT）相比，9月龄DmdMu/Y小鼠的骨骼肌和心肌肌间隙变宽，肌纤维大小不一，有少量炎症细胞浸润（见箭头）；n=3，比例尺为100 μm］；E，检测小鼠肌张力的悬挂实验示意图；F，3月龄和9月龄小鼠前肢悬挂分数统计图（3月龄的DmdMu/Y小鼠n=6，WT小鼠n=5；9月龄的DmdMu/Y小鼠n=8，WT小鼠n=5， *P<0.05，**P<0.01）。

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].

图4 流式细胞法检测 LPS诱导急性炎症模型中DmdMu/Y小鼠血液中性粒细胞和单核粒细胞比例变化注：A和B，3月龄DmdMu/Y小鼠注射脂多糖（LPS）0 h和72 h后的中性粒细胞比例和单核细胞比例变化；C和D，9月龄DmdMu/Y小鼠注射LPS 0 h和72 h后的中性粒细胞比例和单核细胞比例变化。野生型C57BL/6J小鼠（WT）作为对照。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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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

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