ArticleInternational journal of molecular sciences2015
Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids.
Article in International journal of molecular sciences, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 27 papers.
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Who cites it
27 citing papers in PubMed, 84 citations in OpenAlex.
- Toward transgenic multicellular parasites: CRISPR-Cas12a germline editing ofScience advances · 2026Article
- Gene editing and CRISPR-dependent homology-mediated end joining.Experimental & molecular medicine · 2025Review
- REMOVER-PITCh: microhomology-assisted long-range gene replacement with highly multiplexed CRISPR-Cas9.In vitro cellular & developmental biology. Animal · 2024Article
- Differential Mitochondrial Genome Expression of Three Sympatric Lizards in Response to Low-Temperature Stress.Animals : an open access journal from MDPI · 2024Article
- Review
- The biological principles and advanced applications of DSB repair in CRISPR-mediated yeast genome editing.Synthetic and systems biotechnology · 2023Review
- A versatile genomic transgenesis platform with enhancedFrontiers in bioengineering and biotechnology · 2023Article
- Structural analysis of random transgene integration in CHO manufacturing cell lines by targeted sequencing.Biotechnology and bioengineering · 2022Article
- Rational design and construction of multi-copy biomanufacturing islands in mammalian cells.Nucleic acids research · 2022Article
- A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes.Metabolic engineering · 2021Article
- PTSelect™: A post-transcriptional technology that enables rapid establishment of stable CHO cell lines and surveillance of clonal variation.Journal of biotechnology · 2021Article
- Glycoengineering of Mammalian Expression Systems on a Cellular Level.Advances in biochemical engineering/biotechnology · 2021Article
- Various strategies of effector accumulation to improve the efficiency of genome editing and derivative methodologies.In vitro cellular & developmental biology. Animal · 2020Review
- Limited dishevelled/Axin oligomerization determines efficiency of Wnt/β-catenin signal transduction.eLife · 2020Article
- CRISPR/Cas9-Mediated Integration of Large Transgene into PigG3 (Bethesda, Md.) · 2020Article
- Article
- Article
- Intracellular generation of single-strand template increases the knock-in efficiency by combining CRISPR/Cas9 with AAV.Molecular genetics and genomics : MGG · 2018Article
- Production of knock-in mice in a single generation from embryonic stem cells.Nature protocols · 2017Article
- Advancing chimeric antigen receptor T cell therapy with CRISPR/Cas9.Protein & cell · 2017Review
Corrections and comments
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Authors and funding
6 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.