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The TARGATT™ HEK293 Master Cell Line and Knockin Kit was designed for fast and site-specific knockin in HEK293 cells, using an easy-to-use gene knockin approach. The master cell line provided in this kit contains an “attP” integrase-recognition landing pad engineered into the hH11 safe harbor locus in the genome. The kit also contains a cloning plasmid containing a corresponding “attB” sequence into which any gene of interest can be cloned (under control of the CMV promoter or a promoter-of-choice). The expression of the integrase (provided as an integrase plasmid) mediates the stable integration of the transgene into the master cell line (Figure 1). The TARGATT™ integrase technology enables highly efficient (>40% without enrichment and ~90% with selection), and site-specific DNA integration without disruption of internal genes. The TARGATT™ HEK293 master cell line can therefore be used for building mammalian cell libraries with site-specific, single transgene gene knockin and uniform, stable gene expression.

The TARGATT™ HEK293 master cell line and knockin kit are suitable for research applications involving directed-evolution of proteins (vaccine development, drug screening, cell-based gene therapy), genome-wide screening, and other stable cell line generation applications*.

Advantages of using TARGATT™ Master Cell Lines for gene knockin:

  • High efficiency integration
  • Site-specific, stable knockin cell line generation
  • Single copy gene integration into safe harbor locus
  • Gene expression from an active, intergenic locus
  • Low off-target integration

* TARGATT™ master cell lines can be generated in any cell line including stem cells. Please contact Applied StemCell for TARGATT™ cell line engineering services to generate a master cell line in a specific cell line of choice or to build your cell libraries.

This product is for research purposes only.

Please also see our frequently asked question section in our webpage for more details.

Contents:

The cell line and reagents provided in this kit are sufficient for 9 transfections (3 triplicate transfections) according to the given protocol:

  • TARGATT™ HEK293 Master Cell line
  • TARGATT™ 24 CMV-MCS-attB; Cloning Plasmid
  • TARGATT™ 25 mCherry-attB (No Promoter); Positive Control Plasmid
  • TARGATT™ CMV-integrase Plasmid

Additional positive control (TARGATT™ 24 mCherry-attB) and integrase (TARGATT™ CMV-integrase) plasmids are available for purchase for customers who have purchased the kit.

Schematic Representation of the Transgene Integration in the TARGATT™ Master Cell Line

schematic-ast-1305-targatt-hek293

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).

Confirmation of site-specific CMV-MCS plasmid integration

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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.

mCherry expression after transfection and blasticidin enrichment

landingpage-ast-1305-mcherryki-3

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.

Publication: TARGATT™ Master Cell Line

  • Chi, X., Zheng, Q., Jiang, R., Chen-Tsai, R. Y., & Kong, L. J. (2019). A system for site-specific integration of transgenes in mammalian cells. PLOS ONE, 14(7), e0219842.
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