最先端研究セミナー
講演者： 原 孝彦（東京都医学総合研究所 基礎医学研究分野 プロジェクトリーダー）
Title: All-in-one conditional knockout system in ES cells for the functional analysis of stem-cell-related genes
日時： 2021年６月９日（水）12:00- 13:00
※ Zoom開催。URLはMoodleの「HIGO最先端セミナー」にてご確認ください。
Abstract:
We established a novel all-in-one cKO system, which enables highly efficient generation of cKO cells and simultaneous gene modifications, including epitope tagging and reporter gene knock-in. We applied this system to mouse ES cells and generated RNA helicase Ddx1 cKO ES cells. The targeted cells displayed endogenous promoter-driven EGFP and FLAG-tagged DDX1 expression, and they were converted to Ddx1 KO via FLP recombinase. By utilizing this system, we isolated highly pure Ddx1F/F and Ddx1-/- ES cells and found that loss of Ddx1 caused rRNA processing defects, thereby activating the ribosome stress-p53 pathway. We also demonstrated cKO of other genes in ES cells and homologous recombination-non-proficient human HT1080 cells. The frequency of cKO clones was remarkably high for both cell types and reached up to 96% when EGFP-positive clones were analyzed. This all-in-one cKO system will be a powerful tool for rapid and precise analyses of gene functions.
Key publications:
1. Nucl. Acid Res., 2021(in press), doi: 10.1093/nar/gkaa1296
2. Exp. Cell Res., 390: 111915, 2020
3. Cancer Sci., 109: 2479-2489, 2018
4. PLoS ONE, 11: e0157187, 2016
担当分野：組織幹細胞 古賀（内線：6592）
※詳細はこちらから
