利用CRISPR/Cas9技术对VSX 2绿色荧光报告基因人诱导多能干细胞系的构建

Generation of VSX2 fluorescent reporter human induced pluripotent stem cells by CRISPR/Cas9 technology

目的构建表达视觉系统同源框2(VSX 2)增强绿色荧光蛋白(eGFP)报告基因的人诱导多能干细胞系(hiPSCs)。方法根据成簇规律间隔短回文重复序列及其相关蛋白9(CRISPR/Cas9)技术原理,设计VSX 2的小向导RNA(sgRNA),构建敲除质粒及包含左右同源臂的P2A-eGFP供体质粒;2个质粒经测序鉴定后电转野生BC1-hiPSCs细胞系,采用嘌呤霉素筛选获得单克隆,扩增培养并鉴定。采用PCR和Sanger测序鉴定基因型;采用核型分析法分析报告细胞系核型;采用STR法排除细胞交叉污染;采用免疫荧光法及逆转录PCR法检测报告细胞系的多能干细胞分子标志物的表达;通过体外三胚层形成实验鉴定报告细胞系向三胚层分化的能力;应用3D视网膜类器官诱导技术获得视网膜类器官,并采用免疫荧光染色法评价VSX2蛋白与eGFP的共定位情况。结果电转后通过PCR鉴定和Sanger测序成功筛选到1株含有VSX 2-eGFP报告基因的hiPSC系。核型分析结果显示该报告细胞系核型正常。STR鉴定结果表明,BC1-VSX 2 eGFP-iPSCs无细胞系交叉污染。逆转录PCR检测和免疫荧光染色表明,BC1-VSX 2 eGFP-iPSCs中iPSCs标志物基因和蛋白表达阳性,包括NANOG、OCT4、SOX2、DNMT3B和GDF3 mRNA以及NANOG、OCT4、SSEA4和TRA-1-60蛋白。免疫荧光染色表明,由BC1-VSX 2 eGFP-iPSCs分化的三胚层组织中内胚层标志物甲胎蛋白(AFP)、中胚层标志物α-平滑肌肌动蛋白(α-SMA)和外胚层标志物神经细胞特异性微管蛋白(TUJ1)表达均为阳性;eGFP与VSX2共表达于视网膜类器官的神经视网膜。结论成功构建1株表达VSX 2-eGFP报告基因的hiPSCs,该细胞系可实时指示VSX2蛋白的时空表达变化,为视网膜发育、疾病机制研究及治疗方法评价提供有力工具。

Objective To establish a fluorescent reporter human induced pluripotent stem cell line(hiPSCs)for monitoring the expression of visual system homeobox 2(VSX 2).Methods VSX2_small guide RNA(sgRNA)was inserted into vector PX459 to construct knockout plasmid,and the P2A-eGFP knock-in donor plasmid was conducted at the same time.The two plasmids were transfected into BC1-hiPSCs.Single cell clones were generated after treatment of puromycin.Correct insertion was confirmed by PCR and Sanger sequencing.The isogenicity of the parental and the reporter hiPSCs was confirmed by STR analysis and karyotyping.Pluripotency capacity of the reporter hiPSCs was analysed by reverse trascription PCR and immunofluorescence.Three-germ-layer formation experiment was carried out to analyse the multi-lineage differentiation ability of the reporter hiPSCs.The reporter hiPSCs were further differentiated to obtain three-dimension(3D)retinal organoids,and immunofluorescence was used to identify the co-localization of the enhanced green fluorescent protein(eGFP)and VSX2.Results A VSX 2 eGFP reporter hiPSC clone was successfully obtained by CRISPR/Cas9 technology,which was consistent with the parental hiPSCs(BC1-hiPSCs)in morphology,without any chromosomal aberrations or cell line cross-contamination.Reverse transcription PCR assay and immunofluorescence showed obvious positive expressions of iPSCs markers in BC1-VSX 2 eGFP-iPSCs,including NANOG,OCT4,SOX2,DNMT3B and GDF3 mRNA as well as NANOG,OCT4,SSEA4 and TRA-1-60 protein.Theα-fetoprotein(AFP),α-smooth muscle actin(α-SMA)and neuronal classⅢβ-tubulin(TUJ1)were expressed in endoderm,mesoderm and ectoderm,respeetively,derived from BC1-VSX 2 eGFP-iPSCs,and eGFP and VSX2 were co-stained in the neural retinal layer of 3D retinal organoids derived from BC1-VSX 2 eGFP-iPSCs by immunofluorescence.Conclusions VSX2 fluorescent reporter hiPSCs is successfully generated,which can monitor the temporal and spatial expression changes of VSX2 protein in real time,providing a powerful tool for evaluation of retina development mechanism and cell therapy.