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| Mafb基因敲除小鼠的构建及其尿道下裂表型初步分析 |
| 刘振忞1,2,孔晓燕1,2,沈炼桔1,2,3,龙春兰1,2,3,刘星3,4,5,6*,魏光辉3,4,5,6 |
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(1. 重庆医科大学附属儿童医院儿童泌尿生殖发育与组织工程重庆市重点实验室, 重庆 400014;
2. 重庆医科大学附属儿童医院儿科学重庆市重点实验室, 重庆 400014;
3. 重庆医科大学附属儿童医院儿童发育疾病研究教育部重点实验室, 重庆 400014;
4. 重庆医科大学附属儿童医院泌尿外科, 重庆 400014;
5. 重庆医科大学附属儿童医院儿童发育重大疾病国家国际科技合作基地, 重庆 400014;
6. 重庆医科大学附属儿童医院国家儿童健康与疾病临床医学研究中心, 重庆 400014
*通信作者) |
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| 摘要: |
| 目的 利用成簇规律间隔短回文重复序列（CRISPR）及其相关蛋白9（Cas9）构建v-maf肌筋膜纤维肉瘤癌基因同源物B（Mafb）基因敲除小鼠，初步研究该基因缺失对尿道发育的影响。方法 根据CRISPR/Cas9技术原理构建Mafb基因敲除F0代C57BL/6J小鼠；经PCR及基因测序鉴定阳性的F0代小鼠与野生型C57BL/6J小鼠交配繁殖，获得F1代Mafb基因敲除杂合子（Mafb+/-）小鼠；Mafb+/-小鼠间进行交配繁殖，获得Mafb基因敲除纯合子（Mafb-/-）胎鼠。收集雄性Mafb-/-胎鼠阴茎组织，通过免疫荧光技术检测阴茎组织中Mafb蛋白表达情况及尿道缝处上皮钙黏蛋白（E-cadherin）和α-平滑肌肌动蛋白（α-SMA）表达情况，扫描电子显微镜及石蜡切片H-E染色观察阴茎组织形态。结果 成功构建、繁育Mafb+/-小鼠并得到雄性Mafb-/-胎鼠。免疫荧光检测结果显示雄性Mafb-/-胎鼠阴茎组织中几乎不存在Mafb蛋白表达，且相较于野生型小鼠其尿道缝处E-cadherin蛋白表达明显降低、α-SMA蛋白表达明显增高；扫描电子显微镜及H-E染色显示雄性Mafb-/-胎鼠为尿道下裂表型。结论 成功构建Mafb基因敲除小鼠模型，该基因敲除可导致小鼠出现尿道下裂表型及尿道缝处细胞中E-cadherin和α-SMA的表达差异，为进一步研究该基因在尿道下裂发生机制中的作用奠定了基础。 |
| 关键词: v-maf肌筋膜纤维肉瘤癌基因同源物B 基因敲除 基因型鉴定 尿道下裂 动物模型 |
| DOI：10.16781/j.CN31-2187/R.20210861 |
| 投稿时间：2021-09-01修订日期：2021-12-07 |
| 基金项目:国家自然科学基金面上项目（81970571）. |
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| Construction of Mafb knockout mice and preliminary analysis of hypospadias phenotype |
| LIU Zhen-min1,2,KONG Xiao-yan1,2,SHEN Lian-ju1,2,3,LONG Chun-lan1,2,3,LIU Xing3,4,5,6*,WEI Guang-hui3,4,5,6 |
(1. Chongqing Key Laboratory of Child Urogenital Development and Tissue Engineering, Children's Hospital of Chongqing Medical University, Chongqing 400014, China;
2. Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing 400014, China;
3. Key Laboratory of Child Development and Disorders of Ministry of Education, Children's Hospital of Chongqing Medical University, Chongqing 400014, China;
4. Department of Urology, Children's Hospital of Chongqing Medical University, Chongqing 400014, China;
5. China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Children's Hospital of Chongqing Medical University, Chongqing 400014, China;
6. National Clinical Research Center for Child Health and Disorders, Children's Hospital of Chongqing Medical University, Chongqing 400014, China
*Corresponding author) |
| Abstract: |
| Objective To construct v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (Mafb) knockout mice by clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9), and explore the impact brought by this gene deletion on the urethral development. Methods The F0 Mafb knockout C57BL/6J mice were constructed according to the principle of CRISPR/Cas9 technology. F0 mice identified by polymerase chain reaction and gene sequencing were mated with wild-type C57BL/6J mice to obtain F1 Mafb knockout heterozygous (Mafb+/-) mice. Mafb knockout homozygous (Mafb-/-) fetal mice were obtained by mating between Mafb+/- mice. The penile tissues of male Mafb-/- fetal mice were collected. The expression of Mafb protein in the penile tissues and the expression of epithelial cadherin (E-cadherin) and α-smooth muscle actin (α-SMA) in the urethral seam were detected by immunofluorescence. The histological morphology of the penile tissues was observed by scanning electron microscopy and paraffin section hematoxylin-eosin (H-E) staining. Results The Mafb+/- mice were successfully constructed and bred, and male Mafb-/- fetal mice were obtained. Immunofluorescence showed scarce expression of Mafb protein in the penises of the male Mafb-/- fetal mice; compared with wide type mice, the expression of E-cadherin was downregulated and α-SMA was upregulated in the urethral seam. Scanning electron microscopy and H-E staining showed that the phenotype of male Mafb-/- fetal mice was hypospadias. Conclusion The Mafb knockout mouse model is successfully constructed. The knockout of Mafb can lead to the hypospadias phenotype and change the expression levels of E-cadherin and α-SMA on the urethral seam. This model lays a foundation for further study on the role of Mafb in the pathogenesis of hypospadias. |
| Key words: v-maf musculoaponeurotic fibrosarcoma oncogene homolog B gene knockout genotype identification hypospadias animal models |
