基于CRISPR/Cas9系统构建TIA1基因突变的人诱导多能干细胞系  

Generation of Human Induced Pluripotent Stem Cell Line Carrying TIA1Gene Mutation by CRISPR/Cas9 System

作　　者：卢媛媛 安艳茹 LU Yuanyuan;AN Yanru(Institutes of Physical Science and Information Technology,Anhui University,Hefei 230601,China;BGI-Shenzhen,Shenzhen 518083,China)

机构地区：[1]安徽大学物质科学与信息技术研究院,合肥230601 [2]深圳华大生命科学研究院,深圳518083

出　　处：《中国细胞生物学学报》2023年第9期1351-1361,共11页Chinese Journal of Cell Biology

基　　金：深圳市科技研发基金(批准号:JCYJ20180507183628543)资助的课题。

摘　　要：利用CRISPR/Cas9基因编辑技术构建TIA1-P362L突变的人诱导多能干细胞(iPSCs)系,并使其分化为运动神经元,为研究TIA1突变导致肌萎缩侧索硬化症(ALS)的分子机制及药物筛选提供细胞模型。利用CRISPR在线工具设计sgRNA,将其构建到pX330载体中,扩增左右同源臂并将其构建到供体载体中,接着对该供体载体的靶位点进行突变以获得同源重组的Donor质粒。将sgRNA和Donor质粒同时电转到野生型iPSCs系C11中,经G418抗性筛选获得单克隆,利用PCR和Sanger测序鉴定基因型,采用Western blot鉴定TIA1蛋白表达情况,并用核型分析、免疫荧光染色、定量反转录PCR及拟胚体(EBs)三胚层分化对TIA1-P362L突变的iPSCs系进行多能性鉴定,通过抑制SMAD信号通路诱导iPSCs分化为运动神经元并用特异性抗体验证。该研究成功建立具有TIA1-P362L突变的iPSCs系,该细胞系正常表达TIA1蛋白,具有多能性且能成功分化为运动神经元,对阐明TIA1在ALS疾病中的作用具有重要意义。Using CRISPR/Cas9-mediated genome editing,a human iPSCs(induced pluripotent stem cells)line carrying TIA1-P362L mutation was generated and differentiated into motor neurons,providing a useful cell modelfor studying the molecular mechanisms of ALS(amyotrophic lateral sclerosis)and drug screening.sgRNA was designed using the CRISPR online design tool,and was ligated into the pX330 vector as the targeting plasmid.Meanwhile,left and right arms for homologous recombination were amplified and inserted into a Donor vector,which wasmutated subsequently at target site to construct the Donor plasmid for mutation.The two plasmids were co-transfectedinto the iPSCs line C11.After screened for G418 resistance,single clones were selected and expanded.PCR andSanger sequencing were applied to validate the genotype of the selected clones.The expression of TIA1 was identified by Western blot.Pluripotency of the TIA1-P362L iPSCs line was analyzed by karyotyping,immunofluorescence,quantitative reverse transcription PCR and three-germ-layer differentiation of EBs(embryoid bodies).The TIA1-P362L iPSCs were further differentiated into motor neurons by inhibition of SMAD pathway and immunofluorescencewas used to validate the expression of specific motor neuron markers.The above study demonstrated that an iPSCsline carrying TIA1-P362L mutation was successfully constructed,which had normal pluripotency capacity and couldbe differentiated into motor neurons,providing a resource for elucidating the role of TIA1 in ALS disease.

关 键 词：TIA1 CRISPR/Cas9 肌萎缩侧索硬化症 人诱导多能干细胞 运动神经元 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]