Santiago, Y. et al. Targeted gene knockout in mammalian cells using engineered zinc finger nucleases. Proc. Natl Acad. Sci. USA 105, 5809-5814

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Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 05/2008; 105(15):5809-14. DOI: 10.1073/pnas.0800940105
Source: PubMed


Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural-but imperfect-DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR(-/-) cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2-3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR(-/-) cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production.

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    • "biological and medical studies, including gene function analysis, gene modification, and drug development . Of note, traditional homologous recombinationmediated gene knockout technology is limited by the absence of rapid frequency-guaranteed targeting methods (Santiago et al. 2008). Recently, zinc-finger nuclease-mediated gene editing or transcription activator-like effector nucleases (TALEN) and Cas9 RNA-guide endonuclease technology appears to be receiving increasing attention for gene manipulation; however, targeted disruption of lethal genes remains an unsolved problem (Cho et al. 2013; Gaj et al. 2012, 2013; Sung et al. 2013; Urnov et al. 2005). "
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    ABSTRACT: Gene targeting is a critical tool for construction of disease models. However, the application of traditional homologous recombination-mediated gene knockout technology is limited by the absence of rapid frequency-guaranteed targeting methods. Although conventional small hairpin RNA (shRNA)-mediated gene silencing offers an alternative for gene targeting, its application is frequently compromised by lower expression efficiency via RNA interference compared to gene knockout. Here we provide an efficient gene targeting strategy involving drug-inducible synergistic silencing with multiple shRNA molecules. On induction, the levels of the target proteins decreased to undetectable levels in all the tested stable transgenic mammalian cell lines, including HEK293 and embryonic stem cell-derived progenies carrying shRNA silencing cassettes. In a transgenic mouse model carrying a silencing cassette targeting the rhodopsin gene, short-time inducer treatment was sufficient to ablate the rhodopsin protein in the retina, resulting in similar retinal phenotypic changes as those observed in rhodopsin mutant mice. Therefore, on a broad basis, this inducible shRNA gene targeting strategy offers a true gene knockout alternative comparable to conventional RNA interference approaches. Electronic supplementary material The online version of this article (doi:10.1007/s11248-014-9841-9) contains supplementary material, which is available to authorized users.
    Preview · Article · Oct 2014 · Transgenic Research
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    • "Gene knockout is the ultimate way to investigate gene functions by introducing mutations directly and specifically into targeted genes in the genome. Gene knockout was limited to very few model organisms until recently; however, technological innovations brought by the artificial nucleases such as zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs) have enabled us to knockout genes in various organisms through quite simple approaches (Santiago et al. 2008; Ochiai et al. 2010, 2012; Miller et al. 2011; Watanabe et al. 2012; Ansai et al. 2013, 2014; Suzuki et al. 2013; Hayashi et al. 2014; Hosoi et al. 2014; Kondo et al. 2014; Sakane et al. 2014; Sakuma & Woltjen 2014; Sugi et al. 2014). In Ciona, our group have reported knockout of enhanced GFP (eGFP) gene inserted in the genome with ZFNs, and more recently, target mutagenesis of endogenous genes with TALENs (Kawai et al. 2012; Treen et al. 2014; Yoshida et al. 2014). "
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    ABSTRACT: Knockout of genes with CRISPR/Cas9 is a newly emerged approach to investigate functions of genes in various organisms. We demonstrate that CRISPR/Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan. Short guide RNA (sgRNA) and Cas9 mRNA, when they are expressed in Ciona embryos by means of microinjection or electroporation of their expression vectors, introduced mutations in the target genes. The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on-target site. CRISPR/Cas9-mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases.
    Full-text · Article · Sep 2014 · Development Growth and Regeneration
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    • "For recombinant mAb production, a parental cell line with stably reduced PAM expression is desired. To achieve this, genome alterations using ZFNs are the more appropriate tool, as breaks are created at the genomic DNA level that are stably transferred through the subsequent generations of the cell line [3,17]. Two CHO cell lines were generated, as CHO Der2 and CHO Der3 cells, and they were evaluated for PAM at the genomic DNA and mRNA expression levels. "
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    ABSTRACT: Background During development of recombinant monoclonal antibodies in Chinese hamster ovary (CHO) cells, C-terminal amidated species are observed. C-terminal amidation is catalysed by peptidylglycine α-amidating monooxygenase (PAM), an enzyme known to be expressed in CHO cells. The significant variations between clones during clone selection, and the relatively high content of amidated species (up to 15%) in comparison to reference material (4%), led us to develop a cell line with reduced production of C-terminal amidated monoclonal antibodies using genetic manipulation. Results Initial target validation was performed using the RNA interference approach against PAM, which resulted in a CHO cell line with C-terminal amidation decreased to 3%. Due to the transient effects of small-interfering RNAs, and possible stability problems using short-hairpin RNAs, we knocked-down the PAM gene using zinc finger nucleases. Plasmid DNA and mRNA for zinc finger nucleases were used to generate a PAM knock-out, which resulted in two CHO cell lines with C-terminal amidation decreased to 6%, in CHO Der2 and CHO Der3 cells. Conclusion Two genetically modified cell lines were generated using a zinc finger nuclease approach to decrease C-terminal amidation on recombinant monoclonal antibodies. These two cell lines now represent a pool from which the candidate clone with the highest comparability to the reference molecule can be selected, for production of high-quality and safe therapeutics.
    Full-text · Article · Aug 2014 · BMC Biotechnology
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