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Nucleic Acids Res ; 49(7): e40, 2021 04 19.
Article in English | MEDLINE | ID: covidwho-1050155


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.

DEAD-box RNA Helicases/metabolism , Gene Knockout Techniques/methods , RNA, Ribosomal/metabolism , Animals , Cell Line , Embryonic Stem Cells , Fibroblasts , Gene Expression , Gene Knock-In Techniques , Humans , Mice , Mice, Inbred C57BL , RNA Processing, Post-Transcriptional , Ribosomes/metabolism