Due to the dimerization requirement of the FokI endonuclease, a pair of ZFNs is required to cause a double strand break. This strategy requires two different ZFNs to bind at the target site. Each ZFN recognizes a different 12-18 base pair target sequence, and these target sequences must be separated by 4-7 base pairs to allow formation of the catalytically active FokI dimer. These positional constraints drive a very high degree of specificity.
CSTZFN
CompoZr® Custom Zinc Finger Nuclease (ZFN)
ZFN plasmid and mRNA
Synonym(s):
Zinc Finger Nuclease
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About This Item
UNSPSC Code:
12352200
Note: This is a custom product. For more information on pricing and customization options, please fill out the form below and our Technical Sales Specialist will contact you directly.Request Information
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General description
Zinc Finger Nucleases (ZFNs) are a class of engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations. Double-strand breaks are important for site-specific mutagenesis in that they stimulate the cell′s natural DNA-repair processes, namely homologous recombination and Non-Homologous End Joining (NHEJ). By implementing established, field-proven methods, these processes can be harnessed to generate precisely targeted genomic edits, resulting in cell lines with precise and heritable gene deletions, integrations or modifications.
Application
Functional Genomics/Target Validation
- Creation of gene knockouts in multiple cell lines
- Complete knockout of genes not amenable to RNAi
- Creation of knock-in cell lines with promoters, fusion tags or reporters integrated into endogenous genes
- Creation of cell lines that produce higher yields of proteins or antibodies
The CompoZr Custom ZFN service can be used to target genes from a variety of organisms. Validation of the custom ZFNs is completed in a proxy cell line for human, mouse, rat and hamster (CHO) projects. In this assay, the candidate ZFNs are delivered to the proxy cell line and activity is identified and measured. Activity is measured by amplifying over the ZFN target site and then using a nuclease mismatch enzyme to cut DNA strands that have been modified. ZFN efficiency can be measured based on the densitometry results of this assay. CompoZr Custom ZFN Projects for human, mouse, rat, and hamster (CHO) will have a production timeline of 10 weeks after the ZFN design is approved.
Features and Benefits
- Rapid design, assembly, and validation of a ZFN pair targeting your gene of interest
- Rapid and permanent disruption of, or integration into, any genomic loci
- Mutations made are permanent and heritable
- Works in a variety of mammalian somatic cell types
- Edits induced through a single transfection experiment
- Knockout or knock-in cell lines in as little as two months
- Single or biallelic edits occur in 1-20% of clone population
- No antibiotic selection required for screening
Components
- Best Performing ZFN Pair
- 10 Aliquots of Ready-to-Deliver ZFN Pair in mRNA form
- ZFN Pair in Plasmid Form
- Forward and Reverse Primers that allow for determination of rate of mutation and for screening of individual clones
- Positive Control DNA
- Used to determine a baseline cutting efficiency
Other Notes
To have a ZFN made targeting your gene of interest, please inquire about our
Custom ZFN Service offering HERE
Custom ZFN Service offering HERE
Legal Information
All Zinc Finger Nucleases are sold under license from Sangamo Biosciences. For a copy of the Label License provided with purchase of CompoZr ZFNs, please visit the ZFN Label License page.
CompoZr is a registered trademark of Merck KGaA, Darmstadt, Germany
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Description
Pricing
Storage Class Code
10 - Combustible liquids
Flash Point(F)
Not applicable
Flash Point(C)
Not applicable
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Chun Cheng Andy Chen et al.
Physiological genomics, 45(3), 110-118 (2012-12-20)
The present study employed a zinc-finger nuclease strategy to create heterozygous knockout (KO) rats for the transforming growth factor-β1 (Tgfb1) gene on the Dahl SS/Jr genetic background (TGF-β1(+/-) Dahl S). Intercrossing TGF-β1(+/-) rats did not produce any homozygous KO rats
Bettina Schmid et al.
Proceedings of the National Academy of Sciences of the United States of America, 110(13), 4986-4991 (2013-03-05)
Mutations in the Tar DNA binding protein of 43 kDa (TDP-43; TARDBP) are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43(+) inclusions (FTLD-TDP). To determine the physiological function of TDP-43, we knocked out zebrafish Tardbp and
A Tanaka et al.
Leukemia, 27(8), 1621-1627 (2013-02-16)
Human T-cell leukemia virus type 1 (HTLV-1), which causes adult T-cell leukemia (ATL) in humans, establishes a life-long latent infection. Current therapies are not very effective against HTLV-1-associated disorders. A novel therapeutic approach may help to combat HTLV-1 infection. A
David L Mattson et al.
American journal of physiology. Regulatory, integrative and comparative physiology, 304(6), R407-R414 (2013-02-01)
Hypertension and renal damage in Dahl SS rats are associated with increased infiltrating immune cells in the kidney. To examine the role of infiltrating immune cells in this disease process, a zinc finger nuclease targeting bases 672-706 of recombination-activating gene
Carolyn Bentley et al.
The Biochemical journal, 452(2), 313-320 (2013-03-19)
The mutant forms of KRas, NRas and HRas drive the initiation and progression of a number of human cancers, but less is known about the role of WT (wild-type) Ras alleles and isoforms in cancer. We used zinc-finger nucleases targeting
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What specificity should be expected from a ZFN?
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What is the Department of Transportation shipping information for this product?
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Transportation information can be found in Section 14 of the product's (M)SDS.To access the shipping information for this material, use the link on the product detail page for the product.
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What is a Zinc Finger Nuclease (ZFN)?
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A ZFN is a hybrid molecule that couples the DNA binding domain of a zinc-finger protein with the DNA-cleaving nuclease domain of the restriction endonuclease FokI. The DNA binding motif specified by the zinc fingers directs the ZFN to a specific (targeted) locus in the genome.
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Do the ZFNs remain in the cells after causing desired mutations?
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No. The ZFNs are only expressed transiently but the genetic alteration is permanent. The transient nature of ZFNs also reduces the possibility of off-target effects.
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How has the cleavage ability of my ZFN been tested?
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To test the cleavage ability of the ZFNs we use a mismatch-specific nuclease assay. In this assay, PCR primers are used to amplify across the ZFN cleavage site in genomic DNA extracted from ZFN-treated cells. The resulting PCR products, which are now a mixture of wild type and mutated amplicons, are then denatured and re-annealed. When wild type and mutant alleles of the PCR products re-anneal with each other a mismatch occurs. The fragments are then exposed to a mismatch-specific nuclease and the digested reaction is run on a gel to look for smaller migrating cleavage products that indicate cleaved mismatches. The smaller migrating cleavage products reflect the frequency of mutated alleles and are a measure of in vivo ZFN efficacy. Quantitation of the released fragments allows a calculation of the percentage of mutated genes in the population.
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I have a cell line I created by using a ZFN to knockout a gene. I would like to knockout a second gene in this cell line. Is this possible?
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Yes, this is definitely a possibility. It has been shown that at least 3 different genes could be successively knocked out of the genome in the same cell line.
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Can Zinc Finger Nucleases be synthesized to a specific mitochondrial DNA sequence?
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This is target dependent, however the simple answer is yes- as long as sequence content is fairly diverse with some GC content. There is already literature for targeting of mtDNAs with ZFNs.
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Should I expect off-target effects from my ZFN?
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The ZFN reagents that we provide are unlikely to cause significant offtarget effects. The proprietary algorithm that Sigma uses to design the candidate ZFNs targets only sites that are unique within the genome. This, as well as the use of specially engineered obligate heterodimer FokI cleavage domains (Miller et al., Nature Biotechnology, July 25, 2007), helps guard against off-target effects for the ZFN you receive.
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Will a ZFN insert a gene into my cell line?
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Yes. To achieve targeted gene integration you must co-transfect your ZFN with a repair template, consisting of the gene to be inserted flanked by genomic sequence engineered to match (i.e., be homologous to) the sequence surrounding the intended ZFN cut site. After your ZFN cuts at the site specified by its sequence, the natural mechanism of homology dependent repair (HDR) takes over. The cell will recognize the homologous sequence in the repair template and insert the exogenous gene at the site of ZFN cleavage.
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How is the binding specificity of the ZFN tested?
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The binding specificity of the zinc finger modules in the Sangamo archive have previously been characterized by a number of in-vitro methods (ELISA among others).
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