Cutting edge Seminar

 

Speaker: Takahiko Hara (Project Leader, Stem Cell Project, Tokyo Metropolitan Institute of Medical Science)

Title:  All-in-one conditional knockout system in ES cells for the functional analysis of stem-cell-related genes

 

 

Date&Time:  9 Jun.  (Wed.) 2021, 12:00- 13:00

※This seminar can also be attended through ZOOM. Please check the URL on “HIGO Cutting-Edge Seminar” at Moodle.

https://md.kumamoto-u.ac.jp/course/view.php?id=90416

 

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