CRISPR/Cas9技术介导Crx-iCreERT2红色荧光报告人胚胎干细胞系的构建及其三维视网膜类器官培养  

Construction of Crx-iCreERT2 fluorescent reporter human embryonic stem cells by CRISPR/Cas9 technology and 3D retinal organoid culture

作　　者：杜雨馨 刘依宗 阎飞跃 沈吟[1] Du Yuxin;Liu Yizong;Yan Feiyue;Shen Yin(Eye Center,Renmin Hospital of Wuhan University,Wuhan 430060,China;Medical Research Institute,Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院眼科中心,430060 [2]武汉大学医学研究院,430060

出　　处：《中华实验眼科杂志》2021年第5期388-397,共10页Chinese Journal Of Experimental Ophthalmology

基　　金：国家重点研发计划“政府间国际科技创新合作/港澳台科技创新合作”重点专项项目(2017YFE0103400)。

摘　　要：目的利用CRISPR/Cas9技术构建Crx-iCreERT2红色荧光报告人胚胎干细胞(ESCs)系及其3D视网膜类器官培养。方法对H9细胞系靶位点序列进行PCR扩增并测序验证后,利用CRISPR/Cas9技术设计多条sgRNA并对其进行活性检测,根据活性、特异性等因素选择最合适的sgRNA。经酶切鉴定和测序确认打靶载体构建完成后,将打靶载体电转H9细胞系,在hES-ZLM-001基因Exon4和3’-非翻译区之间终止密码子前插入P2A-tdTomato-P2A-iCreERT2,进行药物筛选、阳性克隆富集。设计引物对目标区域进行PCR扩增并测序,根据测序结果和测序峰图选出纯合去抗性敲进阳性细胞克隆。培养所得1-A07细胞系,通过流式细胞分析法检测OCT4阳性细胞比例,采用细胞爬片免疫荧光染色法观察干细胞标志物SOX2、NANOG和SSEA4表达情况。采用核型分析方法检测细胞核型。应用3D培养技术获得视网膜类器官,于分化后不同时间点行冰冻切片,免疫荧光染色法检测不同种类细胞分子标志物的表达分布情况。结果H9细胞系靶位点序列与Genebank和Ensembl所提供序列一致。根据H9细胞系靶位点序列共设计16条sgRNA,最终选择sgRNA8和sgRNA12作为sgRNA。电转后通过PCR筛选得到4个去抗性敲进阳性克隆,其中1-A07细胞系经流式细胞仪分析OCT4阳性细胞比例约为98.7%,所得细胞系外源性tdTomato-P2A-iCreERT2片段重组位置正确,正常表达干细胞标志物,核型分析结果正常。应用3D培养技术可定向诱导1-A07细胞系分化为表达tdTomato红色荧光的视网膜类器官。分化后30 d,出现BRN3A阳性神经节细胞、CALBINDIN阳性水平细胞、CHAT阳性无长突细胞,分化后45 d,出现RECOVERIN阳性光感受器细胞,分化后90 d出现PKCα阳性双极细胞。神经节细胞分布于视网膜类器官深层,水平细胞、无长突细胞、双极细胞分布于中深层,光感受器细胞主要分布于顶层。结论成功构建Crx-iCreERT2红色荧光报告人ESCObjective To establish Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines using CRISPR/Cas9 technology and 3D retinal organoid culture.Methods The target site sequence of H9 cell line was verified by polymerase chain reaction(PCR).SgRNAs were designed by CRISPR/Cas9 technique and their activity was detected.The most optimal sgRNA was selected according to the factors such as activity and specificity.After identification of the target vectors by restriction enzyme and sequencing,the target vectors were transferred to the H9 cell line by electroporation.P2A-tdTomato-P2A-iCreERT2 was inserted between Exon4 and 3’-untranslated region of hES-ZLM-001 gene.Knockin positive clones were obtained after drug treatment,enrichment of positive clones.Primers were designed to perform PCR on the target region,and homozygous de-resistant knockin positive cell clones were selected according to the sequencing results and peaks.The 1-A07 cell line was cultured,and then flow cytometry for the proportion of OCT4 positive cells,immunofluorescence for three stem cell molecular markers including SOX2,NANOG,SSEA4,karyotype analysis were carried out to confirm whether the 1-A07 cell line could be used for further experiments.Retinal organoids were obtained by three-dimensional(3D)culture technology and the expression of molecular markers was detected by immunofluorescence at different developmental stages of retinal organoids.Results The target site sequence of H9 cell line was consistent with that given by Genebank and Ensembl.Sixteen sgRNAs were designed according to the target site sequence of H9 cell line,and finally sgRNA8 and sgRNA12 were selected.The sgRNAs and recombinant plasmids were transfected into the H9 cell line by electroporation,and four homozygous de-resistant knockin positive cell clones were obtained by PCR.Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines were successfully obtained.In 1-A07 cell line,the proportion of OCT4 positive cells was about 98.7% by flow cytometry,and the express

关 键 词：视网膜类器官 3D培养 胚胎干细胞 光感受器前体细胞 

分 类 号：R774.1[医药卫生—眼科]