All-in-one conditional knockout system in ES cells for the functional analysis of stem-cell-related genes
Takahiko Hara
Stem Cell Project, Tokyo Metropolitan Institute of Medical Science
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.
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