Knockout Rats via Embryo Microinjection of Zinc-Finger Nucleases (original) (raw)
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Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases
Proceedings of the National Academy of Sciences, 2011
Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with high efficiency. Whereas ZFN-mediated gene disruption has been demonstrated in laboratory animals such as mice, rats, and fruit flies, ZFNs have not been used to disrupt an endogenous gene in any large domestic species. Here we used ZFNs to induce a biallelic knockout of the porcine α1,3-galactosyltransferase ( GGTA1 ) gene. Primary porcine fibroblasts were treated with ZFNs designed against the region coding for the catalytic core of GGTA1 , resulting in biallelic knockout of ∼1% of ZFN-treated cells. A galactose (Gal) epitope counter-selected population of these cells was used in somatic cell nuclear transfer (SCNT). Of the resulting six fetuses, all completely lacked Gal epitopes and were phenotypically indistinguishable from the starting donor cell population, illustrating that ZFN-mediated genetic modification did not interfere with the cloning process. Neither off-target cleavage events nor...
PLoS ONE, 2011
Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM + and IgG + B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ,1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.
Zinc-finger nucleases: a powerful tool for genetic engineering of animals
Transgenic Research, 2010
The generation of genetically modified animals or plants with gene-targeted deletions or modifications is a powerful tool to analyze gene function, study disease and produce organisms of economical interest. Until recently, the generation of animals with gene targeted manipulations has been accomplished by homologous recombination (HR) in embryonic stem (ES) cells or cloning through nuclear transfer and has been limited to a few species. Recently, a new technology based on the use of gene-targeted zinc-finger nucleases (ZFNs) was developed and used for the generation of organisms with gene-targeted deletions and/or modifications when combined with HR. ZFNs have been used to generate modified organisms such as plants, Drosophila, zebra fish and rats with gene-targeted mutations. This perspective manuscript is a short review on the use of ZFNs for the genetic engineering of plants and animals, with particular emphasis on our recent work involving rats. We also discuss the application of other targeted nucleases, including homing endonucleases. Microinjection of plasmid or mRNA for ZFNs into rat embryos allowed targeted, rapid, complete, permanent and heritable disruption of endogenous loci. The application of ZFNs to generate gene-targeted knockouts in species where ES cells or cloning techniques are not available is an important new development to answer fundamental biological questions and develop models of economical interest such as for the production of humanized antibodies. Further refinements of ZFN technology in combination with HR may allow knock-ins in early embryos even in species where ES cells or cloning techniques are available.
Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases
Nature Biotechnology, 2008
In most vertebrate model systems direct genomic manipulation at a specific locus is still not feasible. Zinc finger nucleases (ZFNs) provide a system to introduce genomic lesions at a specific site in vertebrate cell lines 1-3. Here, we adapt this technology to create targeted mutations in the zebrafish germline in vivo. ZFNs were engineered that recognize sequences in the zebrafish ortholog of the vascular endothelial growth factor-2 receptor, kdra. Co-injection of mRNAs encoding these ZFNs into 1-cell stage zebrafish embryos led to mutagenic lesions at the target site that were transmitted through the germline with high frequency. Thus, engineered ZFNs can introduce heritable mutations into a vertebrate genome. Importantly, this in vivo gene inactivation approach obviates the need for embryonic stem cell lines and will be applicable to most animal species, especially in cases where early stage embryos are easily accessible. The ability to perform fine genomic manipulation in the mouse has firmly established it as a central vertebrate model system. In this case, targeted gene knockouts and knock-ins have been enabled through the establishment and use of embryonic stem cell lines in which the primary genomic manipulation is performed 4, 5. Similar manipulations in other vertebrate models have largely failed due to the difficulty in establishing comparable cell lines. Thus, an alternative means for generating targeted gene knockouts is essential. ZFNs, which are a chimeric fusion between a Cys 2 His 2 zinc finger protein (ZFP) and the cleavage domain of FokI endonuclease 1, 6 , are powerful tools for genomic manipulation in plants 7, 8 , invertebrates 9, 10 and cell lines 2, 3, 11. The DNA-binding specificity is supplied by the ZFP, which can be engineered to recognize a wide variety of target sequences 12-15 , whereas the cleavage activity is provided by the nuclease domain 1, 6. The nuclease domain requires dimerization for activity, and consequently two ZFNs must assemble on DNA with the appropriate geometry for efficient DNA cleavage 16 (Supplementary Fig. 1a). Double-Correspondence should be addressed to N.D.L.
Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases
We describe the use of zinc-finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), in which targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury (ntl) genes and developed a budding yeast–based assay to identify the most active ZFNs for use in vivo. Injection of ZFN-encoding mRNA into one-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Over half the ZFN mRNA-injected founder animals transmitted disrupted ntl alleles at frequencies averaging 20%. The frequency and precision of gene-disruption events observed suggest that this approach should be applicable to any loci in zebrafish or in other organisms that allow mRNA delivery into the fertilized egg.
Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases
Proceedings of the National Academy of Sciences, 2008
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.
Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases
Genome research, 2014
The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and...
Targeted mutagenesis in zebrafish using customized zinc-finger nucleases
Nature Protocols, 2009
Zebrafish mutants have traditionally been obtained using random mutagenesis or retroviral insertions, methods that cannot be targeted to a specific gene and require laborious gene mapping and sequencing. Recently, we and others have shown that customized zinc finger nucleases (ZFNs) can introduce targeted frame-shift mutations with high efficiency, thereby enabling directed creation of zebrafish gene mutations. Here we describe a detailed protocol for constructing ZFN expression vectors, for generating and introducing ZFN-encoding RNAs into zebrafish embryos, and for identifying ZFN-generated mutations in targeted genomic sites. All of our vectors and methods are compatible with previously described Zinc Finger Consortium reagents for constructing engineered zinc finger arrays. Using these methods, zebrafish founders carrying targeted mutations can be identified within four months.
PLoS ONE, 2012
Zinc finger nucleases (ZFNs) enable precise genome modification in a variety of organisms and cell types. Commercial ZFNs were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. Here we report precise targeted mutagenesis of the mouse genome using Oligomerized Pool Engineering (OPEN) ZFNs. OPEN ZFN can be constructed using publicly available resources and therefore provide an attractive alternative for academic researchers. Two ZFN pairs specific to the mouse genomic locus gt(ROSA26)Sor were generated by OPEN selections and used for gene disruption and homology-mediated gene replacement in single cell mouse embryos. One specific ZFN pair facilitated non-homologous end joining (NHEJ)-mediated gene disruption when expressed in mouse zygotes. We also observed a single homologous recombination (HR)-driven gene replacement event when this ZFN pair was co-injected with a targeting vector. Our experiments demonstrate the feasibility of achieving both gene ablation through NHEJ and gene replacement by HR by using the OPEN ZFN technology directly in mouse zygotes.