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
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