基于一个伴发脑血管狭窄的NF1家系的点突变小鼠建模  

Establishment of mutant mice models based on an NF1 family with cerebrovascular stenosis

作　　者：梁晨思 叶梦 霍丽蓉[1] Liang Chensi;Ye Meng;Huo Lirong(Department of Central Laboratory,the Fuxing Hospital,Capital Medical University,Beijing 100038,China)

机构地区：[1]首都医科大学附属复兴医院中心实验室,北京100038

出　　处：《脑与神经疾病杂志》2021年第7期401-406,共6页Journal of Brain and Nervous Diseases

基　　金：北京市自然科学基金(7182065);北京市西城区优秀人才项目(20160031);北京市西城区拔尖团队项目(201915)。

摘　　要：目的利用CRISPR/Cas9技术构建Nf1基因Q181X定点突变的小鼠模型,为研究该点突变与脑血管狭窄的关系提供实验工具。方法根据Nf1基因点突变的位置和基因组结构设计针对Nf1基因Q181X位点的g RNA,经活性检测后,将有活性的gRNA、Cas9 mRNA以及含有点突变序列的donor DNA通过显微注射到小鼠受精卵中,并移植至假孕鼠体内。获得F0代小鼠后,通过PCR和基因测序技术对Nf1基因进行检测和鉴定。对所获的F0代基因突变阳性小鼠继续繁育后获得F1、F2代目标基因阳性小鼠。结果成功构建Nf1-Q181X基因点突变F0代杂合型小鼠3只,F1代杂合型小鼠2只,F2代杂合小鼠6只,经PCR及基因测序鉴定可稳定遗传Nf1-Q181X点突变。结论 H通过CRISPR/Cas9技术成功获得Nf1基因Q181X点突变小鼠模型,为研究该特定点突变与脑血管异常的关系提供了实验工具。Objective To establish Nf1-Q181X gene mutation mice models using CRISPR/Cas9 gene editing technology so as to provide experimental tools for studying the relationship between Nf1-Q181 X mutation and cerebrovascular stenosis.Methods Guide RNA(gRNA)plasmids were designed and constructed based on the Q181 X loci of Nf1 gene mutation.After activity detection,the active single guide RNA(sgRNA)was transcribed and microinjected into C57 BL/6 mice fertilized eggs together with Cas9 nickase mRNA and donor DNA fragment with point mutation.F0 mice were obtained after embryo transfer into pseudopregnant mice.F0 generation positive mice with the target Nf1 gene mutation were confirmed by PCR and gene sequencing.Then F1 generation mice were obtained by positive F0 generation mice crossed with wild-type C57 BL/6 mice and F2 generation mice were obtained by F1 generation matting with each other.Results Finally three positive F0 generation mice,two positive F1 generation mice and six positive F2 mice with Nf1 gene Q181 X heterozygote mutation were established.Conclusion We successfully established and propagated Nf1 gene Q181X mutant mice models using CRISPR/Cas9 technique,which lay a foundation for further study relationship between the target point mutation and cerebrovascular abnormalities.

关 键 词：Nf1基因突变 脑血管狭窄 CRISPR/Cas9 小鼠模型 

分 类 号：R743.32[医药卫生—神经病学与精神病学]