摘要
目的·通过CRISPR/Cas9n(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 nickase)介导的同源定向修复(homologous-directed repair,HDR)技术在小鼠胚胎干细胞(mouse embryonic stem cell,mESC)的中内胚层关键调控分子T-box转录因子Brachyury(即T基因)末端依次敲入手足口病毒2A(foot-and-mouth disease virus 2A,F2A)和增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)以建立T荧光报告细胞系(mTF2A-EGFP)。方法·首先,针对T基因构建特异性单链导向RNA(single guide RNA,sgRNA)质粒和包含F2A-EGFP的供体质粒。利用电穿孔将这2种质粒递送到mESC E14Tg2a(E14)内,通过HDR在T基因末端插入F2A-EGFP。然后,通过药物筛选和基因测序验证所获得的单克隆细胞,并将其诱导形成类胚体(embryonic body,EB)进行分化。通过荧光显微镜和流式细胞技术分别监测mT-F2A-EGFP细胞克隆在分化前后的荧光信号变化,并进行实时定量聚合酶链反应(real-time quantitative reverse transcription polymerase chain reaction,RT-qPCR)来检测多能性标志基因、中内胚层以及外胚层标志基因的转录水平变化。同时,检测克隆细胞的周期、生长曲线,并利用碱性磷酸酶(alkaline phosphatase,AP)染色检测候选克隆干细胞特性。最后,挑选克隆细胞系T1进行了EB分化。利用流式细胞技术分选出分化细胞群中EGFP荧光表达细胞(EGFP+)和无荧光表达的细胞(EGFP-),并检测各谱系基因的表达情况。结果·EGFP被正确插入到E14细胞的T基因,其荧光强度能正确反映T基因表达水平且未产生明显的不良反应。当T1报告克隆分化时,通过流式细胞技术分选出的包含mT-F2A-EGFP的荧光细胞主要高表达中内胚层标志基因。结论·成功构建携带mT-F2A-EGFP的mESC,可实现对T基因调控程度的快速监测,并实时追踪分化过程中表达T基因标记EGFP的中内胚层细胞。
Objective·To establish a T-box transcription factor Brachyury(T gene)fluorescence reporter cell line,in which foot-and-mouth disease virus 2A(F2A)and enhanced green fluorescent protein(EGFP)were knocked in at the end of mouse T gene(mT-F2A-EGFP)in mouse embryonic stem cells(mESCs)by CRISPR/Cas9n(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 nickase)-mediated homologous-directed repair(HDR)technology.Methods·First of all,the specific single guide RNA(sgRNA)plasmid targeting the sequence near the stop codon of the mouse T gene and the plasmid donor containing F2A-EGFP were constructed.These two plasmids were co-delivered into mESCs E14Tg2a(E14)by electroporation.In this way,the desired fluorescent marker EGFP with self-cleaving peptide F2A were introduced into the end of T gene via HDR.Then,the monoclonal cells,obtained after drug selection and verified by sequencing,were induced for differentiation as embryonic bodies(EB),of which the fluorescence signals of mT-F2A-EGFP were monitored by fluorescence microscope and flow cytometry.These reporter clones were also selected before and after differentiation by real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR),which detected the transcription levels of marker genes determing pluripotency,mesendoderm differentiation or ectoderm differentiation.In addition,the cell cycle and growth curve of these clones were detected.Meanwhile,alkaline phosphatase(AP)staining was used to detect the stem cell characteristics of these candidate clones.Finally,the clone T1 carrying mT-F2A-EGFP was selected for EB differentiation.Flow cytometry was used to sort out EGFP expression cells(EGFP+)and non-EGFP expressing cells(EGFP-)from the EBs comprising multiple lineage cells upon differentiation,of which cell lineage markers were checked by RT-qPCR.Results·EGFP was correctly inserted after the T gene in E14,whose fluorescence intensity reflected the expression level of endogenous T without observed side effects.When the fluorescence reporter clone T1 was differentiated,the EGFP+cells sorted by flow cytometry mainly expressed mesendoderm marker genes.Conclusion·The establishment of mESC line carrying mT-F2A-EGFP can realize rapid monitoring of the degree of T regulation,and track mesendoderm cells expressing T marker,EGFP in real time during differentiation.
作者
王靖怡
王琼
WANG Jingyi;WANG Qiong(Department of Histoembryology,Genetics and Developmental Biology,Shanghai Jiao Tong University School of Medicine,Shanghai Key Laboratory of Reproductive Medicine,Shanghai Frontiers Science Center of Cellular Homeostasis and Human Disease,Shanghai 200025,China)
出处
《上海交通大学学报(医学版)》
CAS
CSCD
北大核心
2023年第4期417-427,共11页
Journal of Shanghai Jiao tong University:Medical Science
基金
国家自然科学基金面上项目(31771512,32070865)。