A novel all-in-one conditional knockout system uncovered an essential role of DDX1 in ribosomal RNA processing.

Teruhiko Suzuki, Eiji Katada, Yuki Mizuoka, Satoko Takagi, Yasuhiro Kazuki, Mitsuo Oshimura, Mayumi Shindo, Takahiko Hara

Nucleic acids research 2021 Apr 19

filter terms:

Generation of conditional knockout (cKO) and various gene-modified cells is laborious and time-consuming. Here, we established an 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 embryonic stem cells (ESCs) and generated RNA helicase Ddx1 cKO ESCs. The targeted cells displayed endogenous promoter-driven EGFP and FLAG-tagged DDX1 expression, and they were converted to Ddx1 KO via FLP recombinase. We further established TetFE ESCs, which carried a reverse tetracycline transactivator (rtTA) expression cassette and a tetracycline response element (TRE)-regulated FLPERT2 cassette in the Gt(ROSA26)Sor locus for instant and tightly regulated induction of gene KO. By utilizing TetFE Ddx1F/F ESCs, we isolated highly pure Ddx1F/F and Ddx1-/- ESCs and found that loss of Ddx1 caused rRNA processing defects, thereby activating the ribosome stress-p53 pathway. We also demonstrated cKO of various genes in ESCs 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. © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

Citation

Teruhiko Suzuki, Eiji Katada, Yuki Mizuoka, Satoko Takagi, Yasuhiro Kazuki, Mitsuo Oshimura, Mayumi Shindo, Takahiko Hara. A novel all-in-one conditional knockout system uncovered an essential role of DDX1 in ribosomal RNA processing. Nucleic acids research. 2021 Apr 19;49(7):e40