Generation of VSX2 fluorescent reporter human induced pluripotent stem cells by CRISPR/Cas9 technology / 中华实验眼科杂志

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 VSX2 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- VSX2 eGFP-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- VSX2 eGFP-iPSCs, and eGFP and VSX2 were co-stained in the neural retinal layer of 3D retinal organoids derived from BC1- VSX2 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.