Next Article in Journal
Effect of Relative Arrangement of Cationic and Lipophilic Moieties on Hemolytic and Antibacterial Activities of PEGylated Polyacrylates
Next Article in Special Issue
CRISPR/Cas9-Mediated Rapid Generation of Multiple Mouse Lines Identified Ccdc63 as Essential for Spermiogenesis
Previous Article in Journal
Shikonin Inhibits the Migration and Invasion of Human Glioblastoma Cells by Targeting Phosphorylated β-Catenin and Phosphorylated PI3K/Akt: A Potential Mechanism for the Anti-Glioma Efficacy of a Traditional Chinese Herbal Medicine
Previous Article in Special Issue
Genome Editing Using Mammalian Haploid Cells
Open AccessArticle

Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

1
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan
2
Department of Chemical Engineering, Faculty of Engineering, Kyushu University, Fukuoka 819-0395, Japan
3
Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
*
Authors to whom correspondence should be addressed.
Academic Editor: Izuho Hatada
Int. J. Mol. Sci. 2015, 16(10), 23849-23866; https://doi.org/10.3390/ijms161023849
Received: 29 August 2015 / Revised: 24 September 2015 / Accepted: 25 September 2015 / Published: 9 October 2015
(This article belongs to the Special Issue Genome Editing)
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. View Full-Text
Keywords: TALEN; gene knock-in; CHO cells; microhomology-mediated end-joining TALEN; gene knock-in; CHO cells; microhomology-mediated end-joining
Show Figures

Figure 1

MDPI and ACS Style

Sakuma, T.; Takenaga, M.; Kawabe, Y.; Nakamura, T.; Kamihira, M.; Yamamoto, T. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids. Int. J. Mol. Sci. 2015, 16, 23849-23866.

Show more citation formats Show less citations formats

Article Access Map by Country/Region

1
Only visits after 24 November 2015 are recorded.
Back to TopTop