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- W2580046404 abstract "Fluorescent reporter gene knock-in induced pluripotent stem cell (iPSC) lines have been used to evaluate the efficiency of differentiation into specific cell lineages. Here, we report a knock-in strategy for the generation of human iPSC reporter lines in which a 2A peptide sequence and a red fluorescent protein (E2-Crimson) gene were inserted at the termination codon of the cone-rod homeobox (Crx) gene, a photoreceptor-specific transcriptional factor gene. The knock-in iPSC lines were differentiated into fluorescence-expressing cells in 3D retinal differentiation culture, and the fluorescent cells also expressed Crx specifically in the nucleus. We found that the fluorescence intensity was positively correlated with the expression levels of Crx mRNA and that fluorescent cells expressed rod photoreceptor-specific genes in the later stage of differentiation. Finally, we treated the fluorescent cells with DAPT, a Notch inhibitor, and found that DAPT-enhanced retinal differentiation was associated with up-regulation of Crx, Otx2 and NeuroD1, and down-regulation of Hes5 and Ngn2. These suggest that this knock-in strategy at the 3'-end of the target gene, combined with the 2A peptide linked to fluorescent proteins, offers a useful tool for labeling specific cell lineages or monitoring expression of any marker genes without affecting the function of the target gene." @default.
- W2580046404 created "2017-02-03" @default.
- W2580046404 creator A5039341329 @default.
- W2580046404 creator A5064922173 @default.
- W2580046404 creator A5073604083 @default.
- W2580046404 date "2017-01-26" @default.
- W2580046404 modified "2023-10-14" @default.
- W2580046404 title "Knock-in strategy at 3′-end ofCrxgene by CRISPR/Cas9 system shows the gene expression profiles during human photoreceptor differentiation" @default.
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- W2580046404 doi "https://doi.org/10.1111/gtc.12472" @default.
- W2580046404 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/28124402" @default.
- W2580046404 hasPublicationYear "2017" @default.
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