論文 - 詳細
| RRC ID | 52073 |
|---|---|
| 著者 | Homma K, Usui S, Kaneda M. |
| タイトル | Knock-in strategy at 3'-end of Crx gene by CRISPR/Cas9 system shows the gene expression profiles during human photoreceptor differentiation. |
| ジャーナル | Genes Cells |
| 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. |
| 巻・号 | 22(3) |
| ページ | 250-264 |
| 公開日 | 2017-3-1 |
| DOI | 10.1111/gtc.12472 |
| PMID | 28124402 |
| MeSH | CRISPR-Cas Systems Cell Differentiation* Cells, Cultured Gene Knock-In Techniques* Homeodomain Proteins / biosynthesis Homeodomain Proteins / genetics* Humans Induced Pluripotent Stem Cells / physiology Photoreceptor Cells, Vertebrate / physiology* Receptors, Notch / metabolism Signal Transduction Trans-Activators / biosynthesis Trans-Activators / genetics* Transcriptome* |
| IF | 1.655 |
| 引用数 | 4 |
| オルトメトリクス指標 |
オルトメトリクス指標項目
|
| 最多言及媒体 | X(Twitter) |
| 各媒体での言及数の合計 | 3 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| ヒト・動物細胞 | 454E2(HPS0077) |