摘要
目的利用CRISPR/Cas9技术构建MMACHC基因c.80A>G(p.Q27R)纯合突变的cblC型甲基丙二酸血症合并同型半胱氨酸血症小鼠模型。方法利用CRIPSR/Cas9基因编辑技术,将小鼠MMACHC基因第80位碱基由碱基A突变为碱基G,在靶位点区域设计单链导向RNA(single-guide RNA,sgRNA),构建打靶载体,将Cas9/sgRNA及打靶载体显微注射到小鼠受精卵中,培育获得F0代小鼠,以小鼠鼠尾鉴定基因型,将点突变阳性F0代小鼠与野生型小鼠交配获得具有稳定基因型的F1代小鼠,F1代小鼠交配繁育获得MMACHC基因c.80A>G纯合突变型小鼠,对纯合型小鼠、杂合型小鼠、野生型小鼠进行血代谢产物甲基丙二酸和总同型半胱氨酸检测。结果获得12只MMACHC基因c.80A>G点突变阳性的F1代小鼠,并进行培育繁殖扩大种群,获得纯合突变型小鼠,纯合型小鼠无早期死亡,其血甲基丙二酸、总同型半胱氨酸水平显著高于杂合型和野生型小鼠(P<0.05),杂合型和野生型小鼠血甲基丙二酸、总同型半胱氨酸水平无差异(P>0.05)。结论利用CRISPR/Cas9技术成功构建MMACHC基因c.80A>G(p.Q27R)纯合突变的cblC型甲基丙二酸血症合并同型半胱氨酸血症小鼠模型。
Objective To establish a mouse model of cblC type methylmalonic acidemia combined with hyperhomocysteinemia carrying c.80A>G(p.Q27R)homozygous mutation of MMACHC gene by using CRISPR/Cas9 technology.Methods Using CRIPSR/Cas9 gene editing technology,the 80th base of the mouse MMACHC gene was mutated from base A to base G.Single-guide RNA(SgRNAs)were designed for the target region and the targeting vector was constructed.Cas9/sgRNA and the vector were microinjected into the fertilized eggs of mice to produce F0 generation mice,and their genotypes were verified by mouse tail DNA analysis.F1 generation mice with stable genotypes were obtained by crossing F0 generation mice with wild-type mice,and homozygous mutant mice were generated by intercrossing F1 generation mice.The blood concentrations of methylmalonic acid and total homocysteine were measured for homozygous mice,heterozygous mice and wild-type mice.Results Twelve F1 mice carrying the c.80A>G point mutation in the MMACHC gene were obtained,and homozygous mutant mice were generated.Homozygous mice did not show early mortality,and their survival time was similar to that of wild-type mice.The blood concentrations of methylmalonic acid and total homocysteine were significantly elevated in homozygous mice compared to heterozygous and wild-type mice(P<0.05),while no significant difference was detected between heterozygous and wild-type mice(P>0.05).Conclusions A mouse model of cblC type methylmalonic acidemia combined with hyperhomocysteinemia carrying c.80A>G(p.Q27R)homozygous mutation in MMACHC gene can be constructed via CRISPR/Cas9 technology.
作者
王薇
毛莹莹
郑萍
程沛迪
张文超
吴小兵
马文豪
陈倩
张学
Wang Wei;Mao Yingying;Zheng Ping;Cheng Peidi;Zhang Wenchao;Wu Xiaobing;Ma Wenhao;Chen Qian;Zhang Xue(Department of Neurology,Children’s Hospital,Capital Institute of Pediatrics,Beijing 100020,China;Beijing Ruicy Gene Therapy Institute For Rare Diseases,Beijing 100176,China;Center for Genetic Medicine,Chinese Academy of Medical Science&Peking Union Medical College,Beijing 100005,China)
出处
《中国医学前沿杂志（电子版）》
CSCD
2023年第6期34-39,共6页
Chinese Journal of the Frontiers of Medical Science(Electronic Version)
基金
国家重点研发项目(2022YFC2703903)
北京亦城合作发展基金会科研项目罕见病相关课题资助公益项目(YJXJ-JZ-2021-0014-19)。
