論文 - 詳細
| RRC ID | 74008 |
|---|---|
| 著者 | Yamazaki K, Matsuo K, Okada A, Uno N, Suzuki T, Abe S, Hamamichi S, Kishima N, Togai S, Tomizuka K, Kazuki Y. |
| タイトル | Simultaneous loading of PCR-based multiple fragments on mouse artificial chromosome vectors in DT40 cell for gene delivery. |
| ジャーナル | Sci Rep |
| Abstract |
Homology-directed repair-mediated knock-in (HDR-KI) in combination with CRISPR-Cas9-mediated double strand break (DSB) leads to high frequency of site-specific HDR-KI. While this characteristic is advantageous for generating genetically modified cellular and animal models, HDR-KI efficiency in mammalian cells remains low. Since avian DT40 cells offer distinct advantage of high HDR-KI efficiency, we expanded this practicality to adapt to mammalian research through sequential insertion of target sequences into mouse/human artificial chromosome vector (MAC/HAC). Here, we developed the simultaneous insertion of multiple fragments by HDR method termed the simHDR wherein a target sequence and selection markers could be loaded onto MAC simultaneously. Additionally, preparing each HDR donor containing homology arm by PCR could bypass the cloning steps of target sequence and selection markers. To confirm the functionality of the loaded HDR donors, we constructed a MAC with human leukocyte antigen A (HLA-A) gene in the DT40 cells, and verified the expression of this genomic region by reverse transcription PCR (RT-PCR) and western blotting. Collectively, the simHDR offers a rapid and convenient approach to generate genetically modified models for investigating gene functions, as well as understanding disease mechanisms and therapeutic interventions. |
| 巻・号 | 12(1) |
| ページ | 21790 |
| 公開日 | 2022-12-16 |
| DOI | 10.1038/s41598-022-25959-9 |
| PII | 10.1038/s41598-022-25959-9 |
| PMID | 36526651 |
| PMC | PMC9758134 |
| MeSH | Animals CRISPR-Cas Systems* / genetics Chromosomes, Artificial Gene Editing Genome Humans Mammals / genetics Mice Polymerase Chain Reaction Recombinational DNA Repair* |
| IF | 3.998 |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | X(Twitter) |
| 各媒体での言及数の合計 | 2 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| ヒト・動物細胞 | Hep G2(RCB1886) DT40(RCB1464) |