Enabling Unprecedented Research in Virtually Any Species Including Rats, Mice,
CompoZr Zinc Finger Nuclease (ZFN) Technology is a novel system for rapid creation of targeted gene knockouts, genomic insertions or gene editing in eukaryotic systems. ZFNs are highly efficient pairs of custom nucleases designed and made by Sigma-Aldrich to target your gene or genomic sequence of interest. ZFNs (mRNA or plasmid formats) are delivered to cells by transfection methods or to embryos by microinjection. Upon cleavage of the target site, endogenous cellular processes are harnessed to produce targeted mutations that result in gene knockout.
Recent Developments in Animals
Recent work has shown that the ZFN-based gene knockout method is highly effective not only in cell lines, but also in embryos for the creation of animal models. Such developments enable targeted genetic engineering of organisms such as rats and zebrafish, where conventional methods of genetic manipulation have been unsuccessful. ZFN-based gene targeting has been proven to work in a wide variety of organisms including rats, mice, rabbits, zebrafish, Drosophila and C. elegans with testing in additional model systems underway.
CompoZr ZFN-based genetic engineering does not require the use of embryonic stem (ES) cells because ZFNs can be injected directly into early stage embryos (see illustration below). This allows targeted gene disruption in a wider spectrum of organisms (i.e. rats and zebrafish) and in much shorter timeframes. Knockout rats and mice can be created in as little as 2-3 months at high efficiencies (monoallelic 10-15%, biallelic 1%) compared to the ES cell method in mice that can take up to 12-18 months. ZFN methods result in efficient germline transmission of targeted genetic mutations without incorporation of foreign DNA sequences.
ZFN Knockout Animal Creation via Microinjection
Knockout model creation using Zinc Finger Nucleases starts with a fertilized single cell embryo (see illustration below). The ZFN, in mRNA form, is microinjected into the nucleus, cytoplasm, or yolk (e.g. zebrafish) where it locates the target sequence and creates a double strand break. ZFNs in plasmid form may also be injected into the nucleus. The double strand break stimulates the cellular process of non-homologous end joining and results in the mis-repair of the DNA sequence. The resulting mutation usually gives rise to a knockout genotype/phenotype. The embryos are then plated or implanted into the foster mother and allowed to divide and grow into whole organisms. At birth, the animals are screened for mutations and the founder animals are identified. Biallelic knockouts are achievable in the founder generation. However, monoallelic knockouts can also be interbred to produce biallelic knockout animals.
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How to Order
CompoZr ZFNs (Product No. CSTZFN-1KT) are considered custom projects and are quoted through your local sales representative. You may start the process by completing online ZFN inquiry form. After discussion of the ZFN project details, you will be provided with pricing and relevant licensing details (if applicable).
For more information, email us at SigmaZFN@sial.com.
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