Applied StemCell’s (ASC) proprietary TARGATT™ technology, enables fast and site-specific, stable integration of large DNA fragments (up to 20 kb) into an intergenic, transcriptionally active safe harbor locus with very high efficiency. The preselected locus is engineered to contain an "attP" integrase recognition landing pad where single-copy gene integration occurs when used in conjunction with an “attB” containing donor plasmid and integrase expression.
The TARGATT™ gene editing platform is versatile and can be used for the development of large fragment knock-in cell lines, bioproduction, and library construction. This technology circumvents problems associated with random integration such as position effect, and gene silencing or instability due to the integration of multiple copies of the transgene.
ASC can accurately and efficiently engineer the necessary landing pad into the cell line of your choice. Ready-to-use TARGATT TM Master Cell Lines (iPSC, HEK293, and CHO) are also available for integration of your gene of interest (GOI) at a preselected locus that has been tested for uniformed, high gene expression. Contact us today to schedule your free consultation!
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Figure 1: TARGATT™ Knock-In Strategy. TARGATT™ technology enables fast and site-specific, stable integration of large DNA fragments (up to 22 kb) into an intergenic, transcriptionally active safe harbor locus. We can engineer an "attP" integrase recognition landing pad at a safe harbor locus. Single-copy gene insertion occurs when it is used in conjunction with an “attB” containing donor plasmid and integrase expression.
Unlike other recombinases, such as Cre or Flp, the TARGATT™ integrase recognizes and recombines at two largely unrelated sites, attP and attB, in terms of their sequences. Once the integrase-mediated integration at attB and attP takes place, two new hybrid sites, attL and attR are created at the junctions. These new sites are unrecognizable by integrase; therefore, integrase reaction is uni-directional. Once the DNA is integrated, it will not be excised, making the integration process highly efficient. With TARGATT™ integrase system, the gene is integrated in exactly the Rosa26 or H11 locus permanently.
Yes. We can generate TARGATT™ master cell lines engineered with the “attP” docking site at safe harbor locus.
A single copy.
Any defined promoters provided by the customer or published in the literature can be used.
Your gene of interest will be specifically inserted at your choice of either of the two well-characterized loci: H11 and Rosa26 (or AAVS1 for cell lines).
To date, the largest DNA fragment we were successfully with is 22 kb. Insertion efficiency appears to decrease with increasing DNA fragment size.
Yes, TARGATT™ system is ideal for gene over-expression. Different promoters, e.g., tissue-specific promoters or ubiquitous promoters, and inducible systems (Tet On/Off, loxP-stop-loxP) can be used for tissue-specific, ubiquitous, or inducible gene expression.
Yes, you can express any reporter genes such as GFP, DsRed, mCherry, LacZ, Luciferase, and others.
Yes. This would be a customized service. We need to first insert the docking attP site into a desired locus using CRISPR/Cas9 and then insert the gene of interest into the attP site using TARGATT™.