Quickly engineer mammalian cell lines for consistent, reliable protein expression, promoter screening, full-length antibody screening, and more
Co-invented by Applied StemCell’s CEO, Ruby Tsai, Ph.D., and our Head of Research and Development, Alfonso Farruggio, Ph.D., TARGATT™ large knock-in technology enables rapid, efficient, and precise integration of large DNA fragments—up to 20 kb in a single reaction, more in nested reactions—into a specific intergenic locus.
Because integration is precise, single copy, and targeted to a defined site, we can avoid the challenges encountered with random integration mechanisms such as position effects, gene silencing, and gene instability due to the integration of multiple transgene copies.
These same features also enable time-saving capabilities such as working with pools, whether you are comparing variants or simply need stable gene expression. While you can also do these things with systems that integrate randomly, it takes more time and effort.
Taken together, these features make TARGATT™ an incredibly versatile tool for stable mammalian cell line development.
Figure 1. TARGATT™ technology delivers efficient integration with consistent expression. We inserted mCherry into the TARGATT™ landing pad at the H11 safe harbor site in HEK293 cells and used flow cytometry to assess mCherry expression in two different pools of integrants.
Our optimized system consistently delivers >90% integration efficiency after drug selection (as much as 50% before drug selection). Unlike CRISPR Cas9, TARGATT™ technology does not rely on the host’s homologous recombination machinery, enabling its use in non-dividing cells.
Expression from the H11 locus is highly consistent, as can be seen in the reproducibly tight clustering of the mCherry signal in Figure 1.
Integration is highly efficient even with inserts of 20 kb. Larger insertions can be accommodated with multiple, nested integrations.
This opens the door to expressing multiple proteins while maintaining desired stoichiometric ratios.
Figure 2. Raw integration efficiency (no drug selection) into HEK293 cells remains high as insert size increases from 5.5 to 8.4 kb.
TARGATT™ technology is highly specific for the corresponding landing pad, resulting in negligible off-target events.
For our service projects, we verify on-target insertion using Sanger sequencing at the insertion junctions, and can provide additional characterization should your project require.
Highly efficient integration into the robust, reliably-expressing H11 locus means you can be confident that your inserted genes will be expressed* and you don’t need to hunt for the optimal clone. These features make TARGATT™ technology compatible with streamlined pool-based studies and automated workflows.
*For constructs that have demonstrated expression in mammalian cells.
Because TARGATT™ integration occurs through a controlled, site-specific mechanism, only a single copy of the DNA insert is incorporated into the genome.
The integration process destroys the attP site in the landing pad rendering the process unidirectional and, thus, highly efficient.
Figure 3. Integration using TARGATT technology is single copy. (A) We co-transfected GFP and mCherry TARGATT™ donor plasmids into CHO cells and imaged the resulting cells using the green channel (B), the red channel (C), and as an overlay (D). The lack of yellow cells in the overlay indicates the absence of cells expressing both GFP and mCherry, demonstrating that genomic insertion only occurs in single copy.
TARGATT™ large knock-in technology accelerates and enables a range of cell engineering applications due to a combination of highly efficient integration and robust, reproducible expression from the H11 locus.
Say goodbye to worrying about getting a good integrant and hunting for the right clone.
Start studies sooner, work more efficiently when you can use pools instead of purified clones.
Run studies in more relevant cell types to save time and improve your chances for success in the clinic.
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