論文 - 詳細
| RRC ID | 67247 |
|---|---|
| 著者 | Sakuma T, Takenaga M, Kawabe Y, Nakamura T, Kamihira M, Yamamoto T. |
| タイトル | Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids. |
| ジャーナル | Int J Mol Sci |
| Abstract |
Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins. |
| 巻・号 | 16(10) |
| ページ | 23849-66 |
| 公開日 | 2015-10-9 |
| DOI | 10.3390/ijms161023849 |
| PII | ijms161023849 |
| PMID | 26473830 |
| PMC | PMC4632728 |
| MeSH | Animals CHO Cells Clustered Regularly Interspaced Short Palindromic Repeats / genetics Cricetinae Cricetulus DNA End-Joining Repair / genetics* Endonucleases / metabolism Gene Knock-In Techniques / methods* Genetic Engineering / methods* Genetic Vectors / genetics Homologous Recombination / genetics* Hypoxanthine Phosphoribosyltransferase / genetics Immunoglobulin Fc Fragments / genetics Immunoglobulin Variable Region / genetics Mutagenesis, Insertional / genetics Plasmids / genetics* Promoter Regions, Genetic / genetics Recombinant Fusion Proteins / genetics* |
| IF | 4.556 |
| 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 | Patent(IFI CLAIMS) |
| 各媒体での言及数の合計 | 20 |
| 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 | CHO-K1(RCB0285) |