NEED? Microinjection and homologous recombination inembryonic stem (ES) cells, are robust but overallinefficient. Only a few percent of the injected eggs giving rise totransgenic animals. sperm-mediated gene transfer remain poorly usedalternative strategies to the classical transgenicmethods.
What Is ZFN Technology? Engineered DNA-binding proteins. Highly targeted double-strand break (DSB) withinthe genome. Manipulation of the genome. Unprecedented ease and precision. Double-strand breaks in DNA at user-specifiedlocations. Double-strand breaks are important for site-specific mutagenesis. Stimulate the cells natural DNA-repair processes.
What are Zinc Finger Nucleases?http://www.sigmaaldrich.com
Each Zinc Finger Nuclease (ZFN) consists of twofunctional domains:a] A DNA-binding domain comprised of a chain of two-finger modules.b] A DNA-cleaving domain comprised of the nucleasedomain of Fok I. Recognizing a unique hexamer (6 bp) sequence ofDNA. Two-finger modules are stitched together to form aZinc Finger Protein, each with specificity of ≥ 24 bp. Highly-specific pair of genomic scissors are created.
Designed to target any gene in any genome. Delivered to the cell as DNA or RNA. ZFN proteins are expressed. Translocate to the nucleus. Bind their target sites with high specificity. FokI nuclease forms its catalytically active dimer. Creates a single, specific double-strand break at theuser-defined locus. Living cells have evolved several methods to repairdouble-strand breaks. Endogenous processes can be harnessed to create geneknockouts or knock-ins.
REPAIR: Non-homologous end joining (NHEJ). Homologous recombination (HR). NHEJ is an imperfect repair system-Insertions or deletions of base pairs.-Creation of a frameshift.-Exon skipping.-Disrupt gene translation.-Knockout gene function. Donor plasmid.-Donor for homology directed repair.-Designed to include transgenes for targeted integration
Recent Developments in Animals Highly effective not only in cell lines, but also inembryos for the creation of animal models. Proven to work in a wide variety of organismsincluding rats, mice, rabbits, zebrafish, Drosophilaand C. elegans. Does not require the use of embryonic stem (ES) cells. Injected directly into early stage embryos. Targeted gene disruption in a wider spectrum oforganisms.
Benefits: Unlimited Species Possibilities—Animals with ES cell method limitations can now be targeted Rapid Animal Engineering—Fastest method for creation of knockout rodents (2-3 months) andother higher eukaryotes Robust Mutation Rate—Achieve up to a 10-15% mutation rate in founder animals Heritable Transmission—Faithful germline transmission of targeted mutations Universal Tool—Move quickly from cell line proof-of-concept studies into animals
ZFN Knockout Animal Creation via Microinjectionhttp://www.sigmaaldrich.com
Using ZFNs to Create Modified Cell Lineshttp://www.sigmaaldrich.com
Target Applications Functional Genomics/Target Validation Creation of gene knockouts in multiple cell lines Complete knockout of genes not amenable to RNAi Cell-based screening Creation of knock-in cell lines with promoters, fusiontags or reporters integrated into endogenous genes Cell Line Optimization Creation of cell lines that produce higher yields ofproteins or antibodies
Commercialization Sigma® has a standard offering of ZFNs forHuman, Mouse, and Rat. ZFNs have recently been shown to produce site-specific gene knockouts in Zebrafish (Doyon etal. Nature Biotechnology May 25, 2008). The ZFNs for this particular application were tested ina yeast proxy system that accurately reflects ZFNactivity in many other cell types. CompoZr™ ZFNs CompoZr® Targeted Integration Kit - AAVS1- Rapid Gene Insertion into the Human Cell Lineof Your Choice
CONCLUSION Highly attractive alternative to ES cell manipulationand nuclear transfer technology. Development of large animal models for humandiseases and xeno-transplantations. Agricultural breeding. Medical research. Wide range of new applications in modifying thegenome of species with which it has, untilrecently, remained very difficult to work.
REFERENCES Zinc finger nuclease technology heralds a new era inmammalian transgenesis.BY- Fabienne Le Provost1, Simon Lillico2, BrunoPasset, Rachel Young, Bruce Whitelaw and Jean-LucVilotte. http://www.sigmaaldrich.com/life-science/zinc-finger-nuclease-technology