Homologous recombination-independent large gene cassette knock-in in CHO cells using TALEN and MMEJ-directed donor plasmids

Tetsushi Sakuma, Mitsumasa Takenaga, Kawabe Yoshinori, Takahiro Nakamura, Masamichi Kamihira, Takashi Yamamoto

Research output: Contribution to journalArticle

34 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)23849-23866
Number of pages18
JournalInternational journal of molecular sciences
Volume16
Issue number10
DOIs
Publication statusPublished - Oct 9 2015

Fingerprint

Gene Knock-In Techniques
ovaries
plasmids
hamsters
Homologous Recombination
Cricetulus
genes
Ovary
Plasmids
Genes
Single-Chain Antibodies
cells
Chromosomes
Clustered Regularly Interspaced Short Palindromic Repeats
chromosomes
Clone cells
DNA
Cells
clone cells
Proteins

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

Cite this

Homologous recombination-independent large gene cassette knock-in in CHO cells using TALEN and MMEJ-directed donor plasmids. / Sakuma, Tetsushi; Takenaga, Mitsumasa; Yoshinori, Kawabe; Nakamura, Takahiro; Kamihira, Masamichi; Yamamoto, Takashi.

In: International journal of molecular sciences, Vol. 16, No. 10, 09.10.2015, p. 23849-23866.

Research output: Contribution to journalArticle

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