Myo1h基因敲除小鼠模型的建立与初步观察

doi:10.13591/j.cnki.kqyx.2022.08.001

摘要/Abstract

摘要： 目的构建Myo1h(Myosin 1H)基因与人同源的第30号外显子敲除模型小鼠(Myo1h小鼠)并验证其敲除效率。方法采用Crispr/Cas9技术构建Myo1h基因敲除 F0(the founder)代嵌合体小鼠,并进行配笼繁殖,经过基因型鉴定后获得纯合F2(the second filial generation,子2代)代实验鼠。利用实时荧光定量PCR技术和免疫印迹技术从mRNA水平和蛋白层面验证敲除效果;利用免疫荧光染色技术,对小鼠髁突区域Myo1h的表达进行观察,从体内验证敲除效果;通过Micro-CT三维重建测量有效下颌骨长度,对F2代小鼠进行表型评估;采用Prism 8.0软件包对数据进行统计学分析。结果实时定量PCR和免疫印迹结果显示,Myo1h基因的mRNA水平显著降低(P<0.05)、蛋白表达降低。免疫荧光染色结果显示,Myo1h小鼠体内脑部、肺部以及髁突组织的Myo1h表达显著降低(P<0.05)。通过Micro-CT和身长测量分析进行表型评估后发现,Myo1h基因敲除后小鼠的有效身长比正常对照组短(P<0.05)。结论本研究成功构建出Myo1h基因敲除小鼠,且该基因敲除小鼠与正常对照相比,身长发育受到抑制影响。随着Myo1h基因对发育影响研究的继续深入,或许能为颅颌面骨的生长发育研究提供新的思路。

关键词: Myo1h基因, 小鼠, 敲除, 基因型鉴定, 生长发育

Abstract: Objective To construct Myo1h gene homologous to human exon 30 knockout model mice (Myo1h mice) and to verify its knockout efficiency. Methods The F0 generation of Myo1h knockout mice was established with Crispr/Cas9 technology and homozygous F2 generation mice were obtained after breeding and genotype identification. Knockout efficiency at mRNA level and protein level was validated by real-time fluorescence quantitative PCR and Western Blot. Immunofluorescence assay was used to observe the expression of Myo1h in the condyle of mice to verify the knockout efficiency in vivo. Phenotypic assessment of F2 generation mice was carried out by measuring effective mandibular length through Micro-CT 3D reconstruction. Prism 8.0 software package was used for statistical analysis. Results Real-time quantitative PCR and western blot results showed that the mRNA level of Myo1h gene was significantly decreased (P<0.05), and protein expression was reduced. Immunofluorescence assay results showed that the expression of Myo1h in brain, lung tissues and condyle of Myo1h mice was significantly decreased (P<0.05). Phenotypic assessment by Micro-CT and length measurement analysis revealed that the effective body length of Myo1h knockout mice was shorter than that of control group (P<0.05). Conclusion In this study, model of Myo1h gene knockout mice is successfully constructed and the body length development of knockout mice is inhibited compared to control group. With further study on developmental effects of the Myo1h gene, it may provide new ideas for study on cranio-maxillofacial bone growth and development.

Key words: Myo1h gene, mouse, knock-out, genotyping, growth and development

中图分类号:

R349.64

温士强, 孙榕榕, 时函, 李永明. Myo1h基因敲除小鼠模型的建立与初步观察[J]. 口腔医学, 2022, 42(8): 673-680.

WEN Shiqiang, SUN Rongrong, SHI Han, LI Yongming. Construction and observation of Myo1h gene knockout mouse model[J]. Stomatology, 2022, 42(8): 673-680.

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