• TARGATT™ Knockin Master Cell Line

TARGATT™ Knock-in Master Cell Line and Knock-in Kit (HEK293, CHO, and iPSC)

The TARGATT™ Master Cell Lines (HEK293, CHO, and iPSC) and transgene knock-in kits are an efficient way to generate stable, isogenic, knock-in cell lines and build large mammalian cell libraries, even large transgenes.

  • High knock-in efficiency: with enrichment (up to 90%) or without (up to 40%)
  • Site-specific: well-defined, active, intergenic safe harbor locus
  • Single copy integration
  • Uniform, high level gene expression
  • Unidirectional integration
  • Overcomes challenges posed by random integration

The TARGATT™ Master Cell Lines are ideal for research in immunocompatible cell therapy & directed-differentiation (iPSCs); gene regulatory elements, protein evolution, antibody/ recombinant protein expression and high-yield bioproduction studies (HEK293 & CHO cells).

Products and Services
Support Materials
FAQs
1. Can the AST-3064 TARGATT™ cloning plasmid be used for generating mammalian cell libraries with the TARGATT™ HEK Master cell line?
How can I design primers to confirm insertion of the gene of interest for the AST-1305 Master Cell Line?
Would you share the sequence of the plasmids used in the AST-1305 kit?
Would I be able to use other TARGATT™ Plasmids such as AST-3050 or AST-3051 with the master cell line?
How is the TARGATT™ HEK293 Master Cell Line different from other technologies for purpose of site-specific integration of transgenes and stable cell line generation?
Can I use Hygromycin or G418 in my cell culture system?
Will the integration of the plasmid backbone into the TARGATT™ HEK293 Cell Line affect expression of the gene of interest (transgene)?
Do you have TARGATT™ Master Cell Lines in other cell line backgrounds in addition to HEK293?
What is the size of the transgene that can be integrated into the TARGATT™ HEK293 Master Cell Line with the specified efficiency?
How does the TARGATT™ HEK293 Master Cell Lines compare to other HEK293 master cell lines and kits using similar site-specific technologies?
How does the TARGATT™ technology compare to nuclease-based (Cas9) and other systems such as Flp, Cre, etc.?
Technical Details

Now Launching! TARGATT™ HEK293 Master Cell Line and Knock-in Kit - A valuable research tool to generate large, "ISOGENIC" mammalian cell libraries very efficiently!

The TARGATT™ Knock-in Master Cell Line and Kit uses integrase-based integration of a transgene into a preselected intergenic and transcriptionally active genomic locus (hROSA26, hH11, hAAVS1 or other safe harbor loci) engineered with an integrase recognition attP docking site or “landing-pad). Applied StemCell provides landing-pad ready TARGATT™ Master Cell Lines and Kits in three cell line backgrounds: HEK293, CHO, and hiPSC. 

The knock-in kit provided with the master cell line includes a TARGATT™ cloning plasmid is used to generate the donor plasmid containing the gene of interest (transgene) and also contains a corresponding integrase-recognition attB sequence. When the donor plasmid is transfected into the cell line, the integrase catalyzes the integration of the transgene at the attP-attB sites. This integration is unidirectional which results in a stably integrated knock-in cell line.

The landing-pad in the master cell line is engineered into a well-defined, transcriptionally active, intergenic locus (safe harbor locus) that enables high level expression of the integrated gene-of-interest, and without disruption of internal genes and gene silencing commonly seen with random integration.

These TARGATT™ technology and master cell lines can be utilized for generating stable and isogenic, knock-in cell lines and libraries for a variety of applications including reporter gene expression, gene knockdown, conditional/ inducible gene expression, gene overexpression, directed-differentiation of iPSCs, inserting an overexpression cassette for generating immune-privileged iPSCs, antibody expression libraries, pilot bioproduction of recombinant protein, and disease modeling.

Advantages of TARGATT™ technology:

  • High efficiency and stringent gene knock-in
  • Site-specific integration into a well-defined safe harbor locus
  • Isogenic knock-in cell lines: one variant - one locus - one cell line
  • Stable gene integration with unidirectional integration
  • Fewer cell counts and library sizes needed required
  • Permits non-viral high-throughput library screens
  • Overcomes challenges such as random insertion, gene silencing, multiple copy gene integration, ablated gene expression.

The TARGATT™ Master Cell Lines and Knock-in Kits combines the scalability, affordability, and ease-of-use of bacterial/ yeast systems and the advantages of using mammalian cells (closer to human environment and post-translational modifications) for efficient and stable gene knock-in into cell lines and for library generation.

Comparing TARGATT™ and existing gene editing technologies for generating stable knock-in cell lines:

technical-targatt-celline-comparisontable

Please note that the TARGATT™ integrase-based knock-in technology requires the master cell line to have a landing pad (docking site) engineered into the cell at the chosen locus to be able to knock-in efficiently.

If you would like, Applied StemCell provides custom service to engineer a landing pad and generate a Master Cell Line in the cell line of your choice. Please inquire for further details.

Applications

Potential Applications include but are not limited to:

Immuno-oncology

  • CAR affinity/efficiency
  • CAR specificity and safety screening
  • “Universal” CAR-T cell
  • Discover novel immune targets, checkpoints

 

Antibody Discovery

  • scFv screening
  • Off-target screening with membrane protein library
  • Bioprocessing/ bioproduction

Protein evolution

  • Enzyme activity and specificity (Cas9, DNA modification enzymes)
  • AAV capsid specificity and efficiency
 
  • Screening for regulatory elements (promoters, splicing regulators), post-transcriptional regulation
  • Receptor identification: Ion Channels; GPCR

Stem Cell Research

  • Directed-differentiation to cell-lineages
  • Immuno-compatible/ universal iPSC
 
  • Non-membrane, non-secretory protein library
  • Off-target screening
  • Mammalian two-hybrid assays
Publications

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 ONE14(7), e0219842.

Transgenic Mouse Book Chapters

Description of the technology

  • Zhu, F., Gamboa, M., Farruggio, A. P., Hippenmeyer, S., Tasic, B., Schüle, B., … Calos, M. P. (2014). DICE, an efficient system for iterative genomic editing in human pluripotent stem cells. Nucleic Acids Research42(5), e34. http://doi.org/10.1093/nar/gkt1290.
  • Tasic, B., Hippenmeyer, S., Wang, C., Gamboa, M., Zong, H., Chen-Tsai, Y., & Luo, L. (2011). Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proceedings of the National Academy of Sciences of the United States of America108(19), 7902–7907. http://doi.org/10.1073/pnas.1019507108.

Commentary, comparison with other transgenic methods

  • Rossant, J., Nutter, L. M., & Gertsenstein, M. (2011). Engineering the embryo. Proceedings of the National Academy of Sciences108(19), 7659-7660.

Tet inducible mice generated by TARGATT™

Advantage of Hipp11 (H11) locus

Applications for TARGATT™ technology

  • Carlson, H. L., & Stadler, H. S. (2019). Development and functional characterization of a lncRNA‐HIT conditional loss of function allele. genesis, e23351.
  • Chande, S., Ho, B., Fetene, J., & Bergwitz, C. (2019). Transgenic mouse model for conditional expression of influenza hemagglutinin-tagged human SLC20A1/PIT1. PloS one14(10), e0223052. doi:10.1371/journal.pone.0223052
  • Hu, Q., Ye, Y., Chan, L. C., Li, Y., Liang, K., Lin, A., ... & Pan, Y. (2019). Oncogenic lncRNA downregulates cancer cell antigen presentation and intrinsic tumor suppression. Nature immunology, 1.
  • Matharu, N., Rattanasopha, S., Tamura, S., Maliskova, L., Wang, Y., Bernard, A., ... & Ahituv, N. (2018). CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency. Science, eaau0629.
  • Chen-Tsai, R. Y. (2019). Using TARGATT™ Technology to Generate Site-Specific Transgenic Mice. In Microinjection (pp. 71-86). Humana Press, New York, NY
  • Barrett, R. D., Laurent, S., Mallarino, R., Pfeifer, S. P., Xu, C. C., Foll, M., ... & Hoekstra, H. E. (2018). The fitness consequences of genetic variation in wild populations of mice. bioRxiv, 383240.
  • Ibrahim, L. A., Huang, J. J., Wang, S. Z., Kim, Y. J., Li, I., & Huizhong, W. (2018). Sparse Labeling and Neural Tracing in Brain Circuits by STARS Strategy: Revealing Morphological Development of Type II Spiral Ganglion Neurons. Cerebral Cortex, 1-14.
  • Kumar, A., Dhar, S., Campanelli, G., Butt, N. A., Schallheim, J. M., Gomez, C. R., & Levenson, A. S. (2018). MTA 1 drives malignant progression and bone metastasis in prostate cancer. Molecular oncology.
  • Jang, Y., Broun, A., Wang, C., Park, Y. K., Zhuang, L., Lee, J. E., ... & Ge, K. (2018). H3. 3K4M destabilizes enhancer H3K4 methyltransferases MLL3/MLL4 and impairs adipose tissue development. Nucleic acids researchhttps://doi.org/10.1093/nar/gky982
  • Tang, Y., Kwon, H., Neel, B. A., Kasher-Meron, M., Pessin, J., Yamada, E., & Pessin, J. E. (2018). The fructose-2, 6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC. Journal of Biological Chemistry, jbc-RA118.
  • Chen, M., Geoffroy, C. G., Meves, J. M., Narang, A., Li, Y., Nguyen, M. T., ... & Elzière, L. (2018). Leucine Zipper-Bearing Kinase Is a Critical Regulator of Astrocyte Reactivity in the Adult Mammalian CNS. Cell Reports, 22(13), 3587-3597
  • Kido, T., Sun, Z., & Lau, Y.-F. C. (2017). Aberrant activation of the human sex-determining gene in early embryonic development results in postnatal growth retardation and lethality in mice. Scientific Reports7, 4113. http://doi.org/10.1038/s41598-017-04117-6.
  • Nouri, N., & Awatramani, R. (2017). A novel floor plate boundary defined by adjacent En1 and Dbx1 microdomains distinguishes midbrain dopamine and hypothalamic neurons. Development144(5), 916-927.
  • Li, K., Wang, F., Cao, W. B., Lv, X. X., Hua, F., Cui, B., ... & Yu, J. M. (2017). TRIB3 Promotes APL Progression through Stabilization of the Oncoprotein PML-RARα and Inhibition of p53-Mediated Senescence. Cancer Cell31(5), 697-710.
  • Jiang, T., Kindt, K., & Wu, D. K. (2017). Transcription factor Emx2 controls stereociliary bundle orientation of sensory hair cells. eLife, 6, e23661.
  • Booze, M. L., Hansen, J. M., & Vitiello, P. F. (2016). A Novel Mouse Model for the Identification of Thioredoxin-1 Protein Interactions. Free Radical Biology & Medicine99, 533–543. http://doi.org/10.1016/j.freeradbiomed.2016.09.013.
  • Feng, D., Dai, S., Liu, F., Ohtake, Y., Zhou, Z., Wang, H., ... & Hayat, U. (2016). Cre-inducible human CD59 mediates rapid cell ablation after intermedilysin administration. The Journal of clinical investigation, 126(6), 2321-2333.
  • Sun, N., Yun, J., Liu, J., Malide, D., Liu, C., Rovira, I. I., … Finkel, T. (2015). Measuring in vivo mitophagy. Molecular Cell60(4), 685–696. http://doi.org/10.1016/j.molcel.2015.10.009.
  • Devine, W. P., Wythe, J. D., George, M., Koshiba-Takeuchi, K., & Bruneau, B. G. (2014). Early patterning and specification of cardiac progenitors in gastrulating mesoderm. eLife3, e03848. http://doi.org/10.7554/eLife.03848.
  • Fogg, P. C. M., Colloms, S., Rosser, S., Stark, M., & Smith, M. C. M. (2014). New Applications for Phage Integrases. Journal of Molecular Biology426(15), 2703–2716. http://doi.org/10.1016/j.jmb.2014.05.014.
  • Chen-Tsai, R. Y., Jiang, R., Zhuang, L., Wu, J., Li, L., & Wu, J. (2014). Genome editing and animal models. Chinese science bulletin59(1), 1-6.
  • Park, K.-E., Park, C.-H., Powell, A., Martin, J., Donovan, D. M., & Telugu, B. P. (2016). Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins. International Journal of Molecular Sciences17(6), 810. http://doi.org/10.3390/ijms17060810.
  • Guenther, C. A., Tasic, B., Luo, L., Bedell, M. A., & Kingsley, D. M. (2014). A molecular basis for classic blond hair color in Europeans. Nature Genetics46(7), 748–752. http://doi.org/10.1038/ng.2991.
  • Villamizar, C. A. (2014). Characterization of the vascular pathology in the acta2 r258c mouse model and cerebrovascular characterization of the acta2 null mouse. UT GSBS Dissertations and These (Open Access)Paper 508 (2014)
Have Questions?

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