Applied StemCell’s proprietary site-specific, integrase-based TARGATT™ technology can be used to generate stable, knock-in cells lines with large transgenes including stem cells, very efficiently and quickly. Knock-in is mediated by the integrase at a pre-engineered “docking site” in an intergenic, transcriptionally active genomic locus (safe harbor locus) for high level gene expression without disruption of internal genes. This technology allows only a single-copy integration with very high efficiency with or without clonal selection.
Use our TARGATT™ technology to generate your ”Master” cell lines, reporter/tag lines, for iPSC generation, conditional gene expression models and more.
Also, try our ready-to-use TARGATT™ Master Cell Lines to knock-in transgenes in your own lab.
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Applied StemCell’s proprietary TARGATT™ technology allows for the site-specific integration of large DNA fragments. The TARGATT™ technology uses a serine integrase to mediate an irreversible recombination between a pre-engineered “attP” docking-site and an attP recognition-sequence on the donor vector “attB”, for stable gene integration. The attP docking sites can be engineered into a preselected locus, such as an intergenic, transcriptionally active safe harbor locus, using CRISPR/Cas9. The irreversible transgene integration mediated by the integrase ensures a high efficiency and stability of integration.
Applied StemCell is now providing a TARGATT™ master knock-in cell line generation service
Figure 1. Strategy to knock-in attP "docking sites" into hiPSC Rosa26 locus to generate a TARGATT™ iPSC Master Cell Line
Figure 2. Clone 3 shows heterozygous insertion of attP sequence.
Figure 3. Sequencing of clone C3 shows insertion of 70 bp attP docking site in Rosa26 locus (red)
Figure 4. Schematic illustration of the integrase based knock-in in hiPS cells
Figure 5. PCR Gel electrophoresis to confirm insertion of 5.6 kb fragment in TARGATT™ iPS Master cell line.
Figure 6. PCR to confirm gene knock-in into Rosa26 locus in TARGATT™ Master iPSC line.
We use CRISPR/Cas9 to generate master cell lines by inserting TARGATT™ attP sites at a desired locus.
3-5 months, depending on the nature of the cell line.
So far we have successfully inserted a transgene of 20kb using this method.
We ship at least 2 vials, each at 0.5x10^6 cells/vial, cryopreserved cells per clone with a report of the project. Additional clone(s) and vial(s) are available upon request. For fee-for service projects, you can also have the CRISPR and TARGATT™ vectors upon request.
We have several ready-to-use safe harbor loci to choose from. They are Rosa26, H11, and AAVS1. Docking site insertion can be customized to other loci base on your project.
Currently, we have human iPSC and mouse C57/BL6 ESC and iPSC lines. CHO cells will be launched soon. We are happy to discuss for other cell lines as Custom Cell Line Services.