Now Available! Targeted Integration into the Rat Genome at the Rosa26 Locus
CompoZr Targeted Integration Kits are designed to rapidly integrate a user-specified gene of interest (GOI) into preferred sites of integration in either the human, mouse or rat genome. In the human genome the adeno-associated virus (AAVS1) locus on human chromosome 19 is the preferred site, while the Rosa26 locus is located on chromosome 6 in the mouse and chromosome 4 in the rat. The well characterized Zinc Finger Nucleases (ZFNs) in these kits target each site with high specificity, creating a double strand break. ZFN treated cells then use a pZDonor plasmid with homology arms to either the AAVS1 or Rosa26 genomic regions to replace the natural locus by the repair process of homology directed repair. The pZDonor plasmid can be engineered to contain your GOI and will direct targeted integration of your GOI into the AAVS1 or Rosa26 loci. CompoZr Targeted Integration Kits provide a common integration site within a defined locus to generate stable, uniform gene expression levels.
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||CompoZr Targeted Integration Kit - rRosa26
||CompoZr Targeted Integration Kit - mRosa26
||CompoZr Targeted Integration Kit - AAVS1
||CompoZr pZDonor - AAVS1 Puromycin Vector Kit
Effective Integration Efficiency
- Any human, mouse or rat cell type can be modified with CompoZr Targeted Integration Kits
- Targeted Integration Kit ZFNs are well-validated pairs that cleave the target DNA with high specificity
- ZFN mediated targeted integration can be achieved with high success rates
No Transgene Landing Pads Required
- Prior to ZFN transfection cell lines don’t require engineering of transgene landing pads
- Lack of need for transgene landing pads enables immediate transfection of cell lines with GOI
Rapid Biallelic Integrations with a Single Transfection
- Monoallelic and biallelic integrations have been generated with ZFNs
- Gene integrations can occur in as little as 48 hours
Flexibility of Use
- Expression may be driven by the endogenous promoter or by inserting your promoter of choice
- Puromycin selection is available with the Targeted Integration Kit – AAVS1
CompoZr Targeted Integration Kit Components
- 10 aliquots of single use, ready-to-deliver ZFNs in mRNA format
- pZDonor plasmid
- Forward Genotyping Primer
- Reverse MCS Primer
- Integration Control
- Positive control for PCR reaction
- Used to determine integration efficiency
Schematic of the CompoZr Targeted Integration Kits
A. Your Gene of Interest construct (GOI) is subcloned into the supplied pZDonor plasmid at the multiple cloning site (MCS). The GOI construct consists of any promoter of your choice, the gene and the polyA sequence. The MCS is flanked by left and right homology arms to the genomic integration site.
B. The modified pZDonor now contains your GOI between the homology arms.
C. The modified pZDonor and supplied mRNA, encoding ZFNs that target the genomic integration site of are co-transfected into the cell line of your choice.
D. The ZFNs bind and cut the target site in the nucleus, creating a double strand break. The cell uses the modified pZDonor plasmid as a repair template.
E. ZFN stimulated homology-directed repair leads to targeted integration of your gene construct into the genomic locus in the cell line of your choice. Your gene can be transcribed either through the endogenous promoter or by inserting a promoter of your choice upstream of the GOI.
F. Single cell cloning is used to isolate clones with targeted integration into the target locus.
Zinc Finger Nuclease References
- Inami, Y. et al. 2011. Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells. J. Cell Bio. Advance Online Publication.
- Ginjala, V. et al. 2011. BMI1 is recruited to DNA breaks and contributes to DNA damage induced H2A ubiquitation and repair. Mol. Cell Biol. Advance Online Publication.
- Zou, J. et al. 2011. Oxidase deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease mediated safe harbor targeting. Blood. Advance Online Publication.
- Dekelver, R.C. et al. 2010. Functional genomics, proteomics and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. Genome Res. 20(8):1133-42.
- Hockemeyer, D. et al. 2009. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat. Biotechnol., 27(4):851-7.
- Li, H. et al. 2011. In vivo genome editing restores haemostatis in a mouse model of haemophilia. Nature. Advance Online Publication.
- Cui, X. et al. 2011. Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat. Biotechnol. 29(1):64-7.
- Carbery, ID. et al. 2010. Targeted genome modification in mice using zinc-finger nucleases. Genetics. 186(2):451-9.
- Meyer, M. et al. 2010. Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. Proc. Natl. Acad. Sci. USA. 107(34):15022-6.
View the complete list of Zinc Finger Nuclease References
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