Suzuki Teruhiko, Katada Eiji, Mizuoka Yuki, Takagi Satoko, Kazuki Yasuhiro, Oshimura Mitsuo, Shindo Mayumi, Hara Takahiko
Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
Nucleic Acids Res. 2021 Apr 19;49(7):e40. doi: 10.1093/nar/gkaa1296.
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.
条件性基因敲除（cKO）和各种基因修饰细胞的产生既费力又耗时。在此，我们建立了一个一体化的cKO系统，该系统能够高效地产生cKO细胞并同时进行基因修饰，包括表位标签和报告基因敲入。我们将该系统应用于小鼠胚胎干细胞（ESC），并产生了RNA解旋酶Ddx1 cKO ESC。靶向细胞显示出内源性启动子驱动的EGFP和FLAG标签的DDX1表达，并且它们通过FLP重组酶转化为Ddx1基因敲除细胞。我们进一步建立了TetFE ESC，其在Gt(ROSA26)Sor位点携带一个反向四环素反式激活因子（rtTA）表达盒和一个四环素反应元件（TRE）调控的FLPERT2盒，用于即时和严格调控的基因敲除诱导。通过利用TetFE Ddx1F/F ESC，我们分离出了高度纯化的Ddx1F/F和Ddx1-/- ESC，并发现Ddx1的缺失导致rRNA加工缺陷，从而激活核糖体应激-p53途径。我们还证明了ESC和同源重组缺陷的人HT1080细胞中各种基因的cKO。两种细胞类型的cKO克隆频率都非常高，当分析EGFP阳性克隆时，频率高达96%。这个一体化的cKO系统将成为快速精确分析基因功能的有力工具。
