Neuritin耳蜗支持细胞条件性敲除转基因小鼠模型的构建及鉴定

Construction and identification of neuritin conditional knockout mice in supporting cells of the cochlea

目的应用Cre-LoxP重组酶系统构建Neuritin耳蜗支持细胞特异性敲除的Sox2-CreER:Neuritin^(flox/flox)转基因小鼠模型,初步探讨敲除Neuritin对小鼠耳蜗听功能的影响。方法采用Cre-LoxP重组酶系统,将Neuritin^(flox/flox)小鼠和Sox2-CreER工具鼠进行饲养和繁殖,对其子鼠提取尾组织DNA,采用PCR方法鉴定小鼠基因型,获取基因型Sox2-CreER:Neuritin^(flox/flox)即为耳蜗支持细胞条件性敲除Neuritin的转基因小鼠。对其进行听功能检测,选择听力正常的Sox2-CreER:Neuritin^(flox/flox)小鼠作为实验小鼠,连续3 d腹腔注射tamoxifen诱导CreER发挥作用后,分别于诱导后第4和7天,检测Neuritin敲除小鼠的听力变化,并通过组织免疫荧光检测其耳蜗支持细胞中Neuritin的表达量,进一步验证Sox2-CreER:Neuritin^(flox/flox)小鼠模型的构建效果。结果成功构建耳蜗支持细胞条件性敲除Neuritin基因小鼠模型,该小鼠存活且有繁殖能力。听功能测试结果显示在耳蜗支持细胞内特异性敲除Neuritin基因的小鼠听力阈值与对照组相比明显上升(P<0.05),但都处于正常听力范围内。耳蜗细胞组织形态与野生型相似,免疫荧光结果显示Sox2-CreER:Neuritin^(flox/flox)小鼠耳蜗内Neuritin的表达量明显低于对照组。结论应用Cre-LoxP重组酶系统成功构建了耳蜗支持细胞中条件性敲除Neuritin基因的Sox2-CreER:Neuritin^(flox/flox)转基因小鼠模型,为在动物水平研究Neuritin在听力损失的可能恢复机制中的作用提供一定的研究平台基础。

Objective To knock out neuritingene in cochlear supporting cells by CRISPR/Cas9 technology, and to preliminarily explore the effect of neuritinon cochlear auditory function in mice. Methods The Nuritinflox/flox mice and Sox2-CreER tool mice were reared and bred. DNA of their offspring was extracted from the tail tissues, and mice genotypes were identified by PCR method. The genotype Sox2-CreER: Neuritinflox/floxmice obtained was neuritinconditional knockout in cochlear supporting cells. Sox2-CreER: Neuritinflox/flox mice with normal hearing were selected as experimental subjects, and tamoxifen was injected intraperitoneally for three consecutive days to activate CreER. The hearing of mice were detected on the 4 th and 7 th days after injection, and the expression of neuritinin cochlear supporting cells was detected by tissue immunofluorescence to further verify the availability of Sox2-CreER: Neuritinflox/flox mouse model. Results A neuritin conditional knockout mouse model in cochlear supporting cells was successfully constructed, and the mouse survived and had the ability to reproduce. The result of auditory function test showed that the hearing threshold of mice with conditional knockout of Neuritin gene in cochlear support cells was significantly higher compared with the control group(P<0.05), but they were all within the normal hearing range. The immunofluorescence result showed that the expression of neuritinin the cochlea of Sox2-CreER: Neuritinflox/flox mice was significantly lower than that of the control group, and their histomorphology was similar to the wild type. Conclusion Neuritin conditional knockout mouse model(Sox2-CreER: Neuritinflox/flox) was successfully construct in cochlear supporting cells by Cre-LoxP recombinase system, which provides a research platform for studying the role of neuritin the possible recovery mechanism of hearing loss at the animal level.