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.
CSTZFN
CompoZr® Custom Zinc Finger Nuclease (ZFN)
ZFN plasmid and mRNA
Synonyme(s) :
Zinc Finger Nuclease
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About This Item
Code UNSPSC :
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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Niveau de qualité
Gamme de produits
CompoZr®
Conditions d'expédition
dry ice
Température de stockage
−70°C
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Catégories apparentées
Description générale
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.
Caractéristiques et avantages
- 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
Composants
- 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
Autres remarques
To have a ZFN made targeting your gene of interest, please inquire about our
Custom ZFN Service offering HERE
Custom ZFN Service offering HERE
Informations légales
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
Produit(s) apparenté(s)
Réf. du produit
Description
Tarif
Code de la classe de stockage
10 - Combustible liquids
Point d'éclair (°F)
Not applicable
Point d'éclair (°C)
Not applicable
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Retrouvez la documentation relative aux produits que vous avez récemment achetés dans la Bibliothèque de documents.
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
Yoshihiro Inami et al.
The Journal of cell biology, 193(2), 275-284 (2011-04-13)
Suppression of autophagy is always accompanied by marked accumulation of p62, a selective autophagy substrate. Because p62 interacts with the Nrf2-binding site on Keap1, which is a Cullin 3-based ubiquitin ligase adapter protein, autophagy deficiency causes competitive inhibition of the
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
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
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
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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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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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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 the best way to deliver the ZFN and the repair template into the cells?
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The ZFN itself can be delivered as a plasmid or it can be transcribed into an mRNA transcript for delivery. We have found both plasmid and mRNA to be effective depending on cell type and, therefore, provide customers with ZFNs in both plasmid and mRNA form to test in their own cell lines. Repair templates are typically delivered into the cell as linear DNA. Regardless of form, mRNA, DNA, or plasmid, we have found that the use of nucleofection or electroporation generally produce the highest DNA cleavage efficiencies. Due to this increase in efficiency, we recommend using an electroporation or nucleofection delivery method if possible. Lipid-based transfection also works in many cases, but may result in lower efficiencies.
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When using Product CSTZFN, CompoZr® Custom Zinc Finger Nuclease, ZFN,do I need to use a selection marker for Targeted Gene Integration?
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In many cases the efficiency of Targeted Gene Integration is >1% - even without selection. So selection markers are often not necessary. However, in certain circumstances, selection might be useful to have, e.g., when working with poorly transfectable cells.
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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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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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Can Zinc Finger Nucleases be cloned in an inducible promoter?
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Yes, though we don't have any examples or data around this. It is effectively a typical expression cassette so there should not be a problem. However, there are several potential outcomes. If a pair of catalytically active ZFN expression cassettes are stably integrated into a genome, any "leaky" expression could result in premature gene editing. Additionally, we should also consider the fact that we have no control over the type of modification incurred at the ZFN target site (assuming no donor is introduced). The outcomes might include small insertions and deletions resulting in frameshift (like we want) or it could result in an in-frame modification that may not necessarily yield functional gene knockout.
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My cell line is very hard to transfect. Can I still use ZFNs?
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Efficiency of delivery is very important. We have had great success with viral delivery of ZFNs, including AdV, AAV and lentivirus. If you have a viral delivery system available, then we can give guidance on how to apply it to the use of ZFNs.
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