• TARGATT™
    • Site-Specific Knock-in Technology
    • Single Copy Gene Insertion at a Safe Harbor Locus
    TARGATT™

TARGATT™ Genome Editing

Applied StemCell’s (ASC) proprietary 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 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 line and animal models, bioproduction, and library construction. This technology circumvents problems associated with random integration such as position effect, and gene silencing or instability due to integration of multiple copies of the transgene.

Advantages of TARGATT™ Technology:

  • Site-specific knock-in at a pre-selected safe harbor locus (no random integration)
  • Single copy gene insertion
  • Large transgene knock-in (up to 22 kb)
  • No internal genes are interrupted
  • Uninformed expression
  • High integration efficiency & High-level expression of the transgene
  • Eliminates position effect

ASC can accurately and efficiently engineer the necessary landing pad into the cell line or animal model of your choice. We provide ready-to-use TARGATT TM Master Cell Lines (iPSC, HEK293, and CHO) for the integration of your gene of interest (GOI) at a preselected locus that has been tested for uniformed, high gene expression. ASC even supplies an assortment of off-the-shelf TARGATTTM mouse and rat models that you can use in your gene function, drug screening, and human disease research. To learn more about TARGATTTM and how you can incorporate the technology into your research, contact us today!

TARGATT™ Platforms

TARGATT™ Master iPSCs for Safe Harbor Knock-in

TARGATT™ iPSCs for Safe Harbor Knock-in

- High-efficiency, unidirectional integration
- Site-specific, single-copy gene knock-in
- Well-characterized Master Cell Line

TARGATT™ iPSCs for Safe Harbor knock-in

TARGATT™ CHO Master Cell lines

TARGATT™ CHO Master Cell Lines

- Site-specific gene insertion
- H11 & ASC2 (A2) safe harbor, transcriptionally active genomic hotspots
-Ideal for bioproduction

CTARGATT™ CHO Master Cell Lines

TARGATT™ HEK293 Master Cell Lines

TARGATT™ HEK293 Master Cell Lines

- High knock-in efficiency
- Site-specific, single-copy gene insertion
- Overcomes challenges posed by random integration

TARGATT™ HEK293 Master Cell Lines

 TARGATT™ Site-Specific Knock-in Cell Line Service

TARGATT™ Site-Specific Knock-in Cell Line Generation Service

- You send us your favorite cell line
- We will insert the attP docking site at a specific loci
- Fast turnaround time: get your cell line in as little as 3 months

TARGATT™ Site-Specific Knock-in Cell Line Generation Service

TARGATT™ iPSC-iNK Platform

TARGATT™ iPSC-iNK Platform

- >40% gene integration efficiency
- Site-specific knock-in
- Transfection by lipofectamine, eliminating viral manufacturing

TARGATT™ iPSC-iNK Platform

TARGATT™ Knock-in Mouse Models

TARGATT™ Knock-in Mouse Models

- High-efficiency integration (up to 65%)
- Site-specific integration
- Germline transmitted F! mice in 5-8 months

TARGATT™ Knock-in Mouse Models

 


Technology Comparision

Comparing TARGATT™ and existing gene editing technologies for stable knock-in cell line development:

TARGATT™ - For Knock-in Cell Lines

Contact Us - TARGATT™ Services

FAQs
Why does the TARGATT™ knock-in system have high efficiency?
Do you have TARGATT™ technology available for Knock-in cell lines?
How many copies of the gene will be inserted into the genome?
What promoters are used to drive gene expression?
What is the specific site that my gene of interest will be integrated into?
What is the maximum size of a gene you can insert? Will the efficiency of your system be affected if the gene is too large?
Can I create models to overexpress a gene of interest?
Can I integrate a reporter gene? What kind of reporter genes do you recommend?
Can I use TARGATT™ system to create transgenic models with tissue-specific gene expression?
Besides H11 and Rosa26, can gene be inserted at other loci?
Products and Services
Technical Details

TARGATT™ Knock-In Schematic

TARGATT™ Knock-In Schematic

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.

Publications

TARGATT™ Unique Technology for Antibody Engineering & ScreeningCre-rat resource for models of human diseases

  • Zhang, H., Zheng, Q., & Chen-Tsai, R. Y. (2021). Establishment of a Cre-rat resource for creating conditional and physiological relevant models of human diseases. Transgenic research30(1), 91–104. https://doi.org/10.1007/s11248-020-00226-7

Book Chapters

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.

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 mice generated by TARGATT™ (and cited/published articles)

  • Lindtner, S., Catta-Preta, R., Tian, H., Su-Feher, L., Price, J. D., Dickel, D. E., ... & Pennacchio, L. A. (2019). Genomic Resolution of DLX-Orchestrated Transcriptional Circuits Driving Development of Forebrain GABAergic Neurons. Cell reports, 28(8), 2048-2063.
  • Wang, T. A., Teo, C. F., Åkerblom, M., Chen, C., Tynan-La Fontaine, M., Greiner, V. J., ... & Jan, L. Y. (2019). Thermoregulation via Temperature-Dependent PGD2 Production in Mouse Preoptic Area. Neuron, 103(2), 309-322.
  • Clarke, B. A., Majumder, S., Zhu, H., Lee, Y. T., Kono, M., Li, C., ... & Byrnes, C. (2019). The Ormdl genes regulate the sphingolipid synthesis pathway to ensure proper myelination and neurologic function in mice. eLife8.
  • 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.
  • 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., Wang, C., Broun, A., Park, Y. K., Zhuang, L., Lee, J. E., ... & Ge, K. (2018). H3. 3K4M destabilizes enhancer epigenomic writers MLL3/4 and impairs adipose tissue development. bioRxiv, 301986. doi:https://doi.org/10.1101/301986
  • 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 Reports22(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.
  • Matharu, N., Rattanasopha, S., Maliskova, L., Wang, Y., Hardin, A., Vaisse, C., & Ahituv, N. (2017). Promoter or Enhancer Activation by CRISPRa Rescues Haploinsufficiency Caused Obesity. bioRxiv, 140426.
  • 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 investigation126(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)
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