A novel all-in-one conditional knockout system uncovered an essential role of DDX1 in ribosomal RNA processing
In: Nucleic Acids Research, Jg. 49 (2021-01-27), S. e40
Online
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Zugriff:
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.
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A novel all-in-one conditional knockout system uncovered an essential role of DDX1 in ribosomal RNA processing
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Autor/in / Beteiligte Person: | Suzuki, Teruhiko ; Takagi, Satoko ; Oshimura, Mitsuo ; Kazuki, Yasuhiro ; Hara, Takahiko ; Mizuoka, Yuki ; Shindo, Mayumi ; Katada, Eiji |
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Zeitschrift: | Nucleic Acids Research, Jg. 49 (2021-01-27), S. e40 |
Veröffentlichung: | Oxford University Press (OUP), 2021 |
Medientyp: | unknown |
ISSN: | 1362-4962 (print) ; 0305-1048 (print) |
DOI: | 10.1093/nar/gkaa1296 |
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