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Int. J. Mol. Sci. 2015, 16(10), 23849-23866; doi:10.3390/ijms161023849

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
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)
View Full-Text   |   Download PDF [3189 KB, uploaded 9 October 2015]   |  

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. View Full-Text
Keywords: TALEN; gene knock-in; CHO cells; microhomology-mediated end-joining TALEN; gene knock-in; CHO cells; microhomology-mediated end-joining
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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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.

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