Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids |
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Authors: | Tetsushi Sakuma Mitsumasa Takenaga Yoshinori Kawabe Takahiro Nakamura Masamichi Kamihira Takashi Yamamoto |
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Affiliation: | 1.Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan; E-Mail: ;2.Department of Chemical Engineering, Faculty of Engineering, Kyushu University, Fukuoka 819-0395, Japan; E-Mails: (Y.K.); (M.K.);3.Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan; E-Mail: |
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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. |
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Keywords: | TALEN gene knock-in CHO cells microhomology-mediated end-joining |
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