• TARGATT™ Knockin Master Cell Line

TARGATT™ Knockin Master Cell Line

Use our TARGATT™ Master Cell Lines (HEK29T or CHO) and kits to generate site-specific knock-in cell lines efficiently (even large transgene). TARGATT™’s safe harbor locus-based gene integration overcomes challenges associated with random integration methods.

  1. Higher knock-in efficiency compared to other gene integration technologies, with or without clonal selection

  2. Fast and simple gene knock-in protocol

  3. Single copy integration is compatible with gene amplification

  4. No integration of bacterial backbone which might reduce gene expression

  5. Uniform, high level gene expression

The TARGATT™ Master Cell Lines are ideal for large isogenic cell library construction and high-yield bioproduction studies.

Also available,  TARGATT™-mESC Master Cell Line and Custom TARGATT™ Site-Specific Knock-in Cell Line Service.

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Application Notes

TARGATT™ System in HEK293T Cells:


Figure 1. Schematic representation of site-specific gene insertion using TARGATT™ Master Cell Lines. The TARGATT™ Master Cell Line is engineered with the attP landing pad (or docking site) at a safe harbor locus (hROSA26/ hH11/ hAAVS1). The TARGATT™ PhiC31 integrase catalyzes an irreversible reaction between the attP sites on genome and the attB sites on the donor vector, resulting in integration of a single copy of the GFP reporter gene (positive control) at the preselected locus.

Figure 2. GFP expression in TARGATT™ HEK293T Master Cell Lines The CAG-GFP vector was used to verify fast knock-in in the TARGATT™-HEK293T Master Cell Line. An enriched GFP signal was shown under fluorescence microscopy. (Left) bright field microscopy. (Right) Immunofluorescence; GFP channel.

Figure 3. TARGATT™-HEK293T Master Cell Line transfected with donor plasmid containing GFP and attB. (a) Parental HEK293T cell line only (control); (b) Parental HEK293T cell line was transfected with donor plasmid containing GFP reporter by random insertion (+GFP); (c) TARGATT™-HEK293T Master Cell Line was transfected with GFP donor plasmid before GCV selection (+GFP); (d) TARGATT™-HEK293T Master Cell Line was transfected with GFP donor plasmid after GCV negative selection to eliminate clones without gene of interest (GFP+GCV).

The data indicates that the TARGATT™-HEK293 Master Cell Line system provides a robust, fast and efficient integration platform for generating a uniform cell population with stable transgene expression. This platform paves the way for homogeneous expression of GOI and subsequent biotherapeutic protein production.

Is the AST-0020 TARGATT™ H11-C57BL6 mouse ES Cell Line available for purchase separately?
Would it be possible to exchange the AST-3060 TARGATT™ 20 (CAG-MCS) cloning plasmid with other TARGATT™ plasmids?

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™ (and cited/published articles)

  • 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., Butt, N. A., Phadatare, P. R., Dholakia, K., Vedula, J., ... & Levenson, A. S. (2018). A novel MTA1 knock-in mouse model for the mechanistic and therapeutic studies of MTA1-driven prostate cancer. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Research, 78(13):Abstract nr 5107.

  • 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)

Technical Details

The TARGATT™ Knockin Master Cell Line Kit uses PhiC31 integrase to insert any gene of interest into a preselected intergenic and transcriptionally active genomic locus (hROSA26, hH11, or hAAVS1 safe harbor locus).

The TARGATT™ technology can be utilized for generating knock-in cell lines and libraries for a variety of applications including reporter gene expression, gene knockdown, conditional/ inducible gene expression, gene overexpression, antibody expression libraries, pilot bioproduction of recombinant protein, and disease modeling.

Comparing TARGATT™ and existing gene editing technologies:

Have Questions?

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