ZINC FINGER NUCLEASE TECHNOLOGY
76 likes23,236 views
Zinc finger nucleases (ZFNs) allow for highly targeted editing of the genome. ZFNs consist of a DNA-binding domain made of zinc finger proteins and a DNA-cleaving domain. The ZFN pair binds to a target site and creates a double-strand break, which the cell repairs through non-homologous end joining or homologous recombination, enabling gene knockouts or targeted changes. ZFNs work in many cell types and animal models, providing a more efficient alternative to traditional transgenic techniques. They have applications in functional genomics, cell line engineering, and animal model generation.
![ Each Zinc Finger Nuclease (ZFN) consists of two
functional domains:
a] A DNA-binding domain comprised of a chain of two-
finger modules.
b] A DNA-cleaving domain comprised of the nuclease
domain of Fok I.
Recognizing a unique hexamer (6 bp) sequence of
DNA.
Two-finger modules are stitched together to form a
Zinc Finger Protein, each with specificity of ≥ 24 bp.
Highly-specific pair of 'genomic scissors' are created.](https://image.slidesharecdn.com/zfnpriyesh-130616175626-phpapp02/85/ZINC-FINGER-NUCLEASE-TECHNOLOGY-5-320.jpg)
ZINC FINGER NUCLEASE TECHNOLOGY
- 2. NEED? Microinjection and homologous recombination in embryonic stem (ES) cells, are robust but overall inefficient. Only a few percent of the injected eggs giving rise to transgenic animals. sperm-mediated gene transfer remain poorly used alternative strategies to the classical transgenic methods.
- 3. What Is ZFN Technology? Engineered DNA-binding proteins. Highly targeted double-strand break (DSB) within the genome. Manipulation of the genome. Unprecedented ease and precision. Double-strand breaks in DNA at user-specified locations. Double-strand breaks are important for site- specific mutagenesis. Stimulate the cell's natural DNA-repair processes.
- 4. What are Zinc Finger Nucleases? http://www.sigmaaldrich.com
- 5. Each Zinc Finger Nuclease (ZFN) consists of two functional domains: a] A DNA-binding domain comprised of a chain of two- finger modules. b] A DNA-cleaving domain comprised of the nuclease domain of Fok I. Recognizing a unique hexamer (6 bp) sequence of DNA. Two-finger modules are stitched together to form a Zinc Finger Protein, each with specificity of ≥ 24 bp. Highly-specific pair of 'genomic scissors' are created.
- 7. 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 the user-defined locus. Living cells have evolved several methods to repair double-strand breaks. Endogenous processes can be harnessed to create gene knockouts or knock-ins.
- 8. 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
- 10. Recent Developments in Animals Highly effective not only in cell lines, but also in embryos for the creation of animal models. Proven to work in a wide variety of organisms including rats, mice, rabbits, zebrafish, Drosophila and 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 of organisms.
- 11. 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) and other 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
- 12. ZFN Knockout Animal Creation via Microinjection http://www.sigmaaldrich.com
- 13. Using ZFNs to Create Modified Cell Lines http://www.sigmaaldrich.com
- 14. Provost et al.
- 15. 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, fusion tags or reporters integrated into endogenous genes Cell Line Optimization Creation of cell lines that produce higher yields of proteins or antibodies
- 16. Commercialization Sigma® has a standard offering of ZFNs for Human, Mouse, and Rat. ZFNs have recently been shown to produce site- specific gene knockouts in Zebrafish (Doyon et al. Nature Biotechnology May 25, 2008). The ZFNs for this particular application were tested in a yeast proxy system that accurately reflects ZFN activity in many other cell types. CompoZr™ ZFNs CompoZr® Targeted Integration Kit - AAVS1 - Rapid Gene Insertion into the Human Cell Line of Your Choice
- 17. CONCLUSION Highly attractive alternative to ES cell manipulation and nuclear transfer technology. Development of large animal models for human diseases and xeno-transplantations. Agricultural breeding. Medical research. Wide range of new applications in modifying the genome of species with which it has, until recently, remained very difficult to work.
- 18. REFERENCES Zinc finger nuclease technology heralds a new era in mammalian transgenesis. BY- Fabienne Le Provost1, Simon Lillico2, Bruno Passet, Rachel Young, Bruce Whitelaw and Jean-Luc Vilotte. http://www.sigmaaldrich.com/life-science/zinc- finger-nuclease-technology