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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CSTZFN
CompoZr® Custom Zinc Finger Nuclease (ZFN)
ZFN plasmid and mRNA
Synonim(y):
Zinc Finger Nuclease
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Informacje o tej pozycji
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
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
Other Notes
- 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
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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Klasa składowania
10 - Combustible liquids
flash_point_f
Not applicable
flash_point_c
Not applicable
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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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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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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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Can every gene be targeted by a ZFN?
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Yes. Using our current library of 2-finger zinc finger modules, we believe we can produce a working ZFN for about every 200 bp on average, making genome wide coverage feasible. We plan to continue updating our library of zinc finger modules to increase the genomic coverage.
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Will a ZFN knockout a gene in my cell line?
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Yes. ZFNs for gene knockout typically target within the ORF of coding exons. The ZFN that is delivered to you for gene knockout will cause a targeted double strand breakage (DSB) in the genome of your cells. After the DNA is cut by the ZFN, the cell repairs the breakage by the natural process of non-homologous end joining (NHEJ). This is an imperfect repair process that usually results in the loss or gain of genetic information (typically tens of base pairs) at the site of the DSB. Due to the mutations caused by ZFN-mediated NHEJ, 1-20% of resulting clones are expected to have both copies of the target gene knocked out.
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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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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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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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