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

Authors: Zheng Dandan,  Wang Yuan,  Zhang Zuming,  Guan Yuanyuan,  Xie Bingbing,  Jin Kangxin,  Xiang Mengqing,  Zhong Xiufeng
DOI: 10.3760/cma.j.cn115989-20200729-00535
Published 2020-10-10

Cite as Chin J Exp Ophthalmol, 2020,38(10): 814-820.

Abstract

Objective

To establish a fluorescent reporter human induced pluripotent stem cell line (hiPSCs) for monitoring the expression of visual system homeobox 2 (VSX2).

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 VSX2eGFP 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-VSX2eGFP-iPSCs, including NANOG, OCT4, SOX2, DNMT3B and GDF3 mRNA as well as NANOG, OCT4, SSEA4 and TRA-1-60 protein.The α-fetoprotein (AFP), α-smooth muscle actin (α-SMA) and neuronal class Ⅲ β-tubulin (TUJ1) were expressed in endoderm, mesoderm and ectoderm, respeetively, derived from BC1-VSX2eGFP-iPSCs, and eGFP and VSX2 were co-stained in the neural retinal layer of 3D retinal organoids derived from BC1-VSX2eGFP-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.

Key words:

CRISPR/Cas9; Visual system homeobox 2 gene; Human induced pluripotent stem cells; Reporter cell line; Retinal organoids; Differentiation

Contributor Information

Zheng Dandan
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong 510600, China
Wang Yuan
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong 510600, China
Zhang Zuming
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong 510600, China
Guan Yuanyuan
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong 510600, China
Xie Bingbing
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong 510600, China
Jin Kangxin
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong 510600, China
Xiang Mengqing
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong 510600, China
Zhong Xiufeng
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong 510600, China
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Updated: October 23, 2020 — 7:56 am