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

International Journal of Molecular Sciences

Tetsushi Sakuma¹  Mitsumasa Takenaga¹  Yoshinori Kawabe²  Takahiro Nakamura³  Masamichi Kamihira²  Takashi Yamamoto¹ 

[1] Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan; E-Mail:;Department of Chemical Engineering, Faculty of Engineering, Kyushu University, Fukuoka 819-0395, Japan; E-Mails:;Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan; E-Mail:

关键词: TALEN;    gene knock-in;    CHO cells;    microhomology-mediated end-joining;   

DOI  :  10.3390/ijms161023849

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.