HEK293 – high-efficiency library construction/screening
With our proprietary TARGATT™ technology for site-specific integration of large DNA fragments, we’ve developed a series of TARGATT™ “master” cell lines, including engineered HEK293 cells. These cell lines are available as part of our user-friendly TARGATT™ knock-in kits, allowing you to quickly knock in your gene of interest (GOI) in your lab. Our TARGATT™ HEK293 cell lines demonstrate high-level of gene knock-in efficiency and are ideal for library construction and screening in mammalian cells.
The TARGATT™ Knockin Master Cell Line and Kit uses integrase-based integration of a transgene into a preselected intergenic and transcriptionally active genomic locus (hH11) pre-engineered with an integrase recognition “attP” docking site or “landing-pad”). The H11 locus is well-defined, transcriptionally active, and located at intergenic region (safe harbor locus/genomic hotspot). This locus enables the high-level expression of the integrated gene-of-interest without disruption of internal genes and gene silencing commonly seen with random integration.
Applied StemCell (ASC) provides landing-pad ready TARGATT™ master cell lines and kits.
The TARGATT™ HEK293 Master Cell Line and Knockin Kit includes a TARGATT™ cloning plasmid that contains an integrase-recognition “attB” sequence and can be used to generate the donor plasmid containing the gene of interest (transgene). When the donor plasmid is transfected into the master cell line along with the integrase expression plasmid (also provided in the kit), the integrase catalyzes the integration of the transgene at the attP-attB sites. This integration is unidirectional which results in a stable integrated knock-in cell line.
Main applications
a. Library construction and screening
The TARGATT™ HEK293 Master Cell Lines and Knockin Kit combines the scalability, affordability, and ease of use of bacterial/yeast systems for library screening. Given its high integration efficiency, the TARGATT™ HEK293 system provides up to 107 to 109 library coverage (similar to bacterial/yeast systems), offering the highest library coverage in mammalian cells on the market. The advantages of using mammalian cells are proper post-translational modifications and other epigenetic modifications that are missing in bacterial or yeast cells.
b. Generate stable TARGATT™ HEK293 for AAV production. The current AAV production uses tri-plasmid co-transfection to transiently express AAV components in HEK293 cells. The yield is very low and costly. Using TARGATT™, we can generate stable HEK293 cells with the cap-rep components inserted in the genome, and the gene of interest will be inserted at a safe harbor genomic locus. We are currently looking for partners to make HEK293 cells for AAV production.
TARGATT™ HEK293 Master Cell Lines:
TARGATT™ HEK293 Library Kits:
Contact Information
Reach out to us directly through our contact page for any inquiries. Our team is ready to assist you with your TARGATT™ HEK293 cell needs.
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Figure 1. Schematic representation of TARGATT™ site-specific transgene integration mediated by integrase. The TARGATT™-HEK Master Cell Line was engineered with the attP landing pad at the hH11 safe harbor locus. The TARGATT™ plasmid containing the integrase recognition site, attB is used to clone the transgene. The integrase catalyzes an irreversible reaction between the attP site in the genome and attB site in the donor vector, resulting in integration of the gene of interest at the selected H11 locus. The cells containing the gene of interest can be enriched using the selection marker (gray box).
Figure 2. PCR gel electrophoresis to confirm the knockin of TARGATT™ 24 CMV-MCS-attB plasmid mediated by the TARGATT™ Integrase plasmid, after transfection into the TARGATT™ HEK293 Master Cell Line. Two sets of primers were used to confirm knockin: Upstream (512 bp) and Downstream primers (464 bp). The Human control primers (777 bp) was also used as a control to check the integrity of the cells and the genomic DNA (gDNA). Negative control (-) represents cells transfected with the TARGATT™ 24 CMV-MCS-attB plasmid and a mutant TARGATT™ integrase plasmid that is deficient for integration.
Figure 3. The mCherry integration into the TARGATT™ HEK293 master cell line. Left: Integration mediated by the integrase 72 hours post-transfection. Cells were transfected with the mCherry positive control plasmid and either the provided TARGATT™ integrase plasmid (+Integrase) or a mutant TARGATT™ integrase plasmid deficient for integration (-Integrase). The mCherry plasmid has no promoter and requires the ubiquitous EF1 promoter in the landing pad after integration to express the reporter gene. The integration efficiency of mCherry knockin into landing pad was >40%, without selection. Right: Blasticidin enrichment of TARGATT™ HEK293 cells with a knocked-in mCherry-blasticidin plasmid. Cell pools (with 20x and 40x split ratio) were enriched in selection medium for 3 weeks (without cell sorting). The enrichment of mCherry was about 90%.
after blasticidin selection. Data represents the mean ± SE of two representative experiments done in triplicates
TARGATT™ Technology For Antibody Discovery and Screening
(March 2021)
*Featured in Informa Connect’s eBook: Antibody
Discovery, Selection & Screening
TARGATT™ Master Cell Line
Transgenic Mouse Book Chapters
Description of the technology
Commentary, comparison with other transgenic methods
Tet inducible mice generated by TARGATT™
Advantage of Hipp11 (H11) locus
Applications for TARGATT™ technology