title: Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids.
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.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan. tetsushi-sakuma@hiroshima-u.ac.jp.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan. x000906z@hiroshima-u.ac.jp.
authors.forename: Mitsumasa
authors.initials: M
authors.lastname: Takenaga
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, Fukuoka 819-0395, Japan. kawabe@chem-eng.kyushu-u.ac.jp.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kawabe
authors.affiliation: Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan. tnaka@agr.kyushu-u.ac.jp.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, Fukuoka 819-0395, Japan. kamihira@chem-eng.kyushu-u.ac.jp.
authors.forename: Masamichi
authors.initials: M
authors.lastname: Kamihira
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan. tybig@hiroshima-u.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
mesh_terms: D000818:Animals; D016466:CHO Cells; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D059766:DNA End-Joining Repair / Q000235:genetics*; D004720:Endonucleases / Q000378:metabolism; D055879:Gene Knock-In Techniques / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D059765:Homologous Recombination / Q000235:genetics*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics*
chemical_list: D007141:Immunoglobulin Fc Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D007041:Hypoxanthine Phosphoribosyltransferase; D004720:Endonucleases
keywords: CHO cells; TALEN; gene knock-in; microhomology-mediated end-joining