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Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids
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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632728/ https://www.ncbi.nlm.nih.gov/pubmed/26473830 http://dx.doi.org/10.3390/ijms161023849 |
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author | Sakuma, Tetsushi Takenaga, Mitsumasa Kawabe, Yoshinori Nakamura, Takahiro Kamihira, Masamichi Yamamoto, Takashi |
author_facet | Sakuma, Tetsushi Takenaga, Mitsumasa Kawabe, Yoshinori Nakamura, Takahiro Kamihira, Masamichi Yamamoto, Takashi |
author_sort | Sakuma, Tetsushi |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4632728 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-46327282015-11-23 Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids Sakuma, Tetsushi Takenaga, Mitsumasa Kawabe, Yoshinori Nakamura, Takahiro Kamihira, Masamichi Yamamoto, Takashi Int J Mol Sci Article 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. MDPI 2015-10-09 /pmc/articles/PMC4632728/ /pubmed/26473830 http://dx.doi.org/10.3390/ijms161023849 Text en © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Sakuma, Tetsushi Takenaga, Mitsumasa Kawabe, Yoshinori Nakamura, Takahiro Kamihira, Masamichi Yamamoto, Takashi Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids |
title | Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids |
title_full | Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids |
title_fullStr | Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids |
title_full_unstemmed | Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids |
title_short | Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids |
title_sort | homologous recombination-independent large gene cassette knock-in in cho cells using talen and mmej-directed donor plasmids |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632728/ https://www.ncbi.nlm.nih.gov/pubmed/26473830 http://dx.doi.org/10.3390/ijms161023849 |
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