基于CRISPR/Cas9n技术建立携带mT-F2A-EGFP报告系统的小鼠胚胎干细胞系

doi:10.3969/j.issn.1674-8115.2023.04.003

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (4): 417-427.doi: 10.3969/j.issn.1674-8115.2023.04.003

• 论著 · 基础研究 • 上一篇

基于CRISPR/Cas9n技术建立携带mT-F2A-EGFP报告系统的小鼠胚胎干细胞系

王靖怡(), 王琼()

上海交通大学基础医学院组织胚胎学与遗传发育学系，上海市生殖医学重点实验室，上海市细胞稳态调控与疾病前沿科学研究基地，上海 200025

收稿日期:2023-01-29 接受日期:2023-03-21 出版日期:2023-04-28 发布日期:2023-04-28
通讯作者: 王琼 E-mail:1459892116@qq.com;wangqiong@shsmu.edu.cn
作者简介:王靖怡（1998—），女，硕士生；电子信箱：1459892116@qq.com。
基金资助:
国家自然科学基金面上项目(31771512)

Establishment of a mouse embryonic stem cell line carrying a reporter of mT-F2A-EGFP based on CRISPR/Cas9n technology

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

Received:2023-01-29 Accepted:2023-03-21 Online:2023-04-28 Published:2023-04-28
Contact: WANG Qiong E-mail:1459892116@qq.com;wangqiong@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(31771512)

摘要/Abstract

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（mT-F2A-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。

关键词: 小鼠胚胎干细胞, CRISPR/Cas9n, 中内胚层分化, 增强绿色荧光蛋白, 报告基因, Brachyury, 手足口病毒2A

Abstract:

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.

Key words: mouse embryonic stem cell (mESC), CRISPR/Cas9n, mesendoderm differentiation, enhanced green fluorescent protein (EGFP), reporter gene, Brachyury, foot-and-mouth disease virus 2A (F2A)

中图分类号:

R394.1

王靖怡, 王琼. 基于CRISPR/Cas9n技术建立携带mT-F2A-EGFP报告系统的小鼠胚胎干细胞系[J]. 上海交通大学学报（医学版）, 2023, 43(4): 417-427.

WANG Jingyi, WANG Qiong. Establishment of a mouse embryonic stem cell line carrying a reporter of mT-F2A-EGFP based on CRISPR/Cas9n technology[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(4): 417-427.

图/表 7

表1 RT-qPCR引物序列

Tab 1 Primer sequences for RT-qPCR

Gene	Forward primer (5'→3')	Reverse primer (5'→3')
Gapdh	CTCCACTCACGGCAAATTCA	CGCTCCTGGAAGATGGTGAT
Sox2	GCGGAGTGGAAACTTTTGTCC	CGGGAAGCGTGTACTTATCCTT
Gsc	TTGCACAGACAGTCGATGCTACT	TCGTTGCTTTCTCGACCCC
T	TCCTCCATGTGCTGAGACTTGT	CCAAGAGCCTGCCACTTTG
Eomes	GCGCATGTTTCCTTTCTTGAG	GGTCGGCCAGAACCACTTC
nestin	CAGGATTGGGAGGAGGGCAGAG	GGAGGCAGGAGACTTCAGGTAG
Pax6	GTTCCCTGTCCTGTGGACTC	ACCGCCCTTGGTTAAAGTCT
Sox1	ATACCGCAATCCCCTCTCAG	ACAACATCCGACTCCTCTTCC
Dux	CCCAGCGACTCAAACTCCTTC	GGACTTCGTCCAGCAGTTGAT
Zscan4	GAGATTCATGGAGAGTCTGACTGATGAGTG	GCTGTTGTTTCAAAAGCTTGATGACTTC
Zscan4d	GTCCTGACAGAGGCCTGCC	GAGATGTCTGAAGAGGCAAT

图1 pX461-mT-sgRNA质粒和mT-F2A-EGFP-Donor质粒的构建Note：A. Scheme of pX461-mT-sgRNA plasmid map. B. Sequencing result of mT-sgRNA cloned into pX461-Cas9n-2A-Hygro plasmid. C. Scheme of mT-F2A-EGFP-Donor plasmid map.

Fig 1 Construction of pX461-mT-sgRNA plasmid and mT-F2A-EGFP-Donor plasmid

图2 共转染pX461-mT-sgRNA和mT-F2A-EGFP-Donor质粒Note： A. The scheme showed that F2A-EGFP was inserted before the stop codon of T gene. B. The cell morphology of E14 cells 24 h after electroporation with 2 kinds of plasmids (upper left), 3 d after Hygro and blasticidin selection (middle upper) and another 3 d with blasticidin (upper right). The untransfected E14 cells were used as a control (bottom).

Fig 2 Co-transfection of pX461-mT-sgRNA and mT-F2A-EGFP-Donor plasmid

图3 mT-F2A-EGFP单克隆细胞的鉴定Note：A. Gel electrophoresis of PCR products with primers of T-HAL-F/T-EGFP-R (left, 1 596 bp ) or T-BSD-F/T-HAL-R (right, 1 435 bp). B. Gel electrophoresis of PCR products with primers of mT-F/mT-R (3 253 bp). C. Sequencing results of T1, T2, T3, T4 and T5 clones. Primers used for PCR were also labeled here. PC—positive control; NC—negative control; M—marker.

Fig 3 Identification of mT-F2A-EGFP clones

图4 mT-F2A-EGFP单克隆细胞分化过程中各标记基因的相对表达量Note： RT-qPCR experiments show relative mRNA levels of pluripotent marker (Sox2), mesendoderm marker (T, Eomes and Gsc), and ectoderm marker (nestin). RNA of E14, T1, T2, T3, T4 and T5 clones were collected before differentiation (D0), and on the 3rd (D3) and the 4th (D4) day of EB differentiation.

Fig 4 Relative expression levels of marker genes during lineage differentiation of mT-F2A-EGFP clones

图5 mT-F2A-EGFP荧光报告干细胞系的功能检测Note：A. Cell morphology and EGFP expression of E14, T1, T3 and T4 clones on Day 0 (D0), Day 3 (D3) and Day 4 (D4) of EB differentiation upon LIF removal (-LIF). All the microscopic pictures were taken under ×10 magnification. B. Flow cytometry analysis of E14, T1, T3 and T4 on D0, D3 and D4 of EB differentiation upon LIF removal (-LIF). Pink indicates EGFP＋ cells, and red indicates EGFP- cells. C. AP staining of E14, T1, T3, and T4 cells. All the microscopic pictures were taken under ×10 magnification. D. The growth curve of E14, T1, T3 and T4. E. Cell cycle analysis of E14, T1, T3 and T4 cells.

Fig 5 Function test of mT-F2A-EGFP clones

图6 应用mT-F2A-EGFP报告系统分选中内胚层细胞Note：A. Flow cytometry analysis of E14 and T1 on EB differentiation day 4. Pink represents EGFP＋ cells, and red represents EGFP- cells. B. Both EGFP＋ and EGFP- cells of T1 on EB differentiation day 4 were collected by flow cytometry for RNA extraction. Relative mRNA levels of pluripotent marker Sox2, ectoderm markers Pax6 and Sox1, 2CLC markers Dux, Zscan4d and Zscan4, and mesendoderm markers T, Eomes and Gsc of EGFP＋ and EGFP- cells were detected by RT-qPCR. ① P=0.000, ②P=0.005, compared with the EGFP- group.

Fig 6 mT-F2A-EGFP fluorescent reporter stem cell line used to sort mesendoderm cell populations

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基于CRISPR/Cas9n技术建立携带mT-F2A-EGFP报告系统的小鼠胚胎干细胞系

Establishment of a mouse embryonic stem cell line carrying a reporter of mT-F2A-EGFP based on CRISPR/Cas9n technology

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