zinc finger nuclease
Post on 29-Nov-2014
Embed Size (px)
Zinc finger nuclease--a new knock-out technique
Why zinc finger nuclease in zebrafish? 1. There is no effectvie reverse genetic tool for targeted knockouts in zebrafish. 2. RNAi and morpholino come with substantial technical limitation.
What is a Zinc Finger Nuclease (ZFN)?
Stephen C. Ekker, 2008
How ZFN works?
Zinc Finger Engineering
combinatorial-based selection method-- Oligomerized Pool ENgineering
How can we use ZFN?
Advantage of Zinc Finger Nuclease 1. Highly specific 2. Available in many organism: zebrafish, human, rat... 3. High efficiency (compared to homologous recombination)
Summary 1. Zinc finger nuclease consists of Zinc finger targeting specific DNA sequences and FokI for cleavage of DNA. 2. Zinc finger nuclease is powerful Knockout technique: -specific target to the gene of interest -high efficiency
Why are these sometimes referred to as ZFN pairs? A pair of ZFNs is required to cleave double-stranded DNA. This is a requirement of the FokI must dimerize to achieve a double strand break in the DNA. You will be provided w for your target site that has been validated to cut at the endogenous chromosomal locu line.
What specificity should be expected from a ZFN? Due to the dimerization requirement of the FokI endonuclease, a pair of ZFNs is require double strand break. This strategy requires two different ZFNs to bind at the target site. recognizes a different 12-18 base pair target sequence, and these target sequences mu by 4-7 base pairs to allow formation of the catalytically active FokI dimer. These positio drive a very high degree of specifi city.
How is mutation induced?-non-homologous end joining
double strand breakage
non-homologous end joining template mutation induced
What are the advantages to delivering ZFNs to cells in mRNA form? There are many advantages to using a ZFN mRNA transcript. We have found the followi
1. Eliminates the risk of random genome integration of the expression plasmid DNA. 2. Lower cytotoxicity (RNA vs. DNA). 3. Higher efficiency in most tested cases. 4. Expanded range of cell types that zinc finger nucleases can be applied to because s 5. Eliminates the need to use different promoters for ZFN expression in certain cell typ 6. Eliminates the necessity of nuclear delivery, allowing a larger range of transfection re 7. Lower off-target events by exposing cells to ZFNs for a shorter time (mRNA has a sh
What is zinc finger protein?
PD Doland, 2006
Zinc Finger Protein-the most abundant DNA binding motif
Early approaches of genome editing
1. genome-wide, non-targeted approaches -radiation -chemical induced mutagenesis 2. conventional gene targeting -homologous recombination -low efficiency (sophisticated strategy)