• CHO and HEK293
    • Antibody discovery/screening
    • High gene expression locus
    CHO and HEK293

TARGATT™ Antibody Screening Library Construction Service & Kits

TARGATT™ technology enables faster and efficient site-specific integration of large DNA fragments in cell lines. Our proprietary technology offers an ideal platform for generating stable cell line libraries for mammalian cell display-mediated antibody engineering, protein evolution screening, mammalian two-hybrid (M2H) screens and more. It allows only a 1:1 variant-to-cell ratio with a uniform and consistent expression of the gene/ protein for efficient screening.

Applied StemCell (ASC) has integrated its TARGATT™ site-specific knockin technology into CHO and HEK293 cells. The TARGATT™ Master CHO and HEK293 Cell Lines enable single-copy insertion at a safe-harbor locus (H11, ASC2). High integration efficiencies and medium to high levels of protein expression have been observed. ASC can work with you to generate the library that best fits your project needs. TARGATT™ CHO-K1 Kits (H11) for antibody library screening are also available for you to build your own library!

TARGATT™ Advantages:

  • Single copy knockin: 1 cell, 1 docking site, 1 inserted transgene
  • Site-specific knockin into a high expression, safe harbor locus (H11, ASC2)
  • High efficiency integration (HEK293: >40% without, >90% with drug selection; CHO: ~18% without, >90% with drug selection)
  • Large cell library construction in HEK293/ CHO cells
  • Cost-effective: no virus packaging time and resources
  • BSL1 compatible
TARGATT Master Cell Lines

TARGATT HEK293 Master Cell Lines

The TARGATT HEK293 Master Cell Lines provide an efficient way to generate site-specific, stable knockin cell lines. 

TARGATT HEK293 Master Cell Lines

TARGATT CHO Master Cell Lines

The TARGATT CHO Master Cell Lines allow site-specific gene insertion and offer a platform for affordable and feasible bioproduction. 

TARGATT CHO Master Cell Lines

______________________________________________________________

TARGATT™ Library Screen Master Cell Lines: HEK293 & CHO


What do current library screening systems lack?
Traditionally, library screening is completed using bacteria or yeast phage display. Bacteria allow for the creation of large libraries in a short period of time while yeast provides a eukaryotic environment. These systems may be cost-effective, but they lack posttranslational modifications that mammalian cells permit.

Mammalian cells also offer a human-like environment, but the currently available systems are slow and laborious to work with at a high cost. The available mammalian library systems for screening may provide an environment closer to the human system, but the coverage they allow is very low compared to bacteria and yeast.

Applied StemCell’s Solution

Our goal was to engineer the TARGATT™ system into HEK293 & CHO cells in order to:

  • Develop a mammalian display system with a higher efficiency
  • Provide a mammalian display system that can reach E. coli and
    yeast library sizes

Figure 1: Comparison of the currently available library screening systems.

TARGATT™ Mammalian Display
To address the current library screening and size issues, Applied StemCell is using its TARGATT™ gene editing technology to develop a mammalian display system that can consistently hit within an order of magnitude typical for bacteria and yeast. When comparing the TARGATT™ system for mammalian display to other available systems, it is clear that the TARGATT™ system offers unique features including site-specific and single-copy gene insertion.

Table 1: A comparison of the TARGATT™ mammalian display with available alternative display systems.

FAQs
What is the difference between AST-1400, AST-1405, AST-1409, and AST-1410?
Could you describe the business model to access your technology?
How long does it take to obtain a pool? Could this be used for high-throughput Ab production?
Can you tell us a bit more about the screening process, does it require clonal selection?
Is the library constructing plasmid available for us to make our own library by ourselves?
What is the difference between the AST-1306/AST-1307 antibody screening kits and the AST-1305 knockin kit?
AST-1306 & AST-1307: How many screenings can we do with these kits?
AST-1306 & AST-1307: Is additional integrase available?
AST-1306 & AST-1307: I want to test this. What is the next step?
AST-1306 & AST-1307: Are you able to make a construct library for the GOI that I have?
Can I use this system in my own cell line?
AST-1409 and 1410: I am interested in the kit, what information can I obtain in order to insert my GOI?
AST-3080 and 3081: Do you disclose the termination site?
AST-3080 and 3081: Do you disclose the promoter, and its sequence?
AST-1409: A plasmid was transfected in by electroporation or lipofectamine?
AST-1409: A plasmid was introduced by lentiviral or other retrovirus methods?
AST-1409: The cell line was modified using CRISPR/Cas9 engineering?
Regarding the TARGATT™ 41 attB-mCherry-P2A-LacZ (library) Cloning Plasmid: After cloning the gene/s of interest into this plasmid using the NEB kit, the protocol recommends transforming using E. cloni® 10G SUPREME SOLOs electrocompetent cells. How would y
Why should I use ASC's HEK293/CHO TARGATT system rather than phage-display or other display methods?
With more copies will I have a higher yield?
In the treatise (PLOS ONE, 14 (7), e0219842.), The GFP positive rate before GCV selection is 12.2% even in HEK, but it is ~ 40% described on the website. Is this due to the difference between the paper and the system?
In the one in the paper, it seems that PhiC is also incorporated in the Host Cell, but is it the type that Kit also introduces CMV-integrase plasmid at the same time? If possible, could you please tell me why you are changing?
There are four types of kits for antibody library screening, but how do you plan to use them properly? Which one is better suited for building as large a library as possible?
You can do up to 10e9, but is it possible to provide a protocol for building a large-scale library?
Is there a recommended protocol for displaying antibodies? It will be helpful if you have information on how to express Hch / Lch and what to anchor.
Information on gene transfer in CHO is shown in Neon, but is there any information in other devices (NEPA, Nucleofactor2b, etc.)?
You can also request the construction of a library, but can you give us an estimate of the price?
Why is TARGATT™ better?
Which TARGATT™ CHO Kit should I use?
Which TARGATT™ CHO Kit should we use if we are not isolating single antibodies (eg. the best binders)?
Support Materials
Technical Details

logo-TARGATT

Applications for High Resolution Protein Screening:

  • Directed evolution (vaccine development, drug screening, cell-based gene therapy)
  • Genome-wide screening
  • Bioproduction/ stable cell line generation

schematic-targatt-protein-evolution

Figure 1. Schematic representation of the workflow involved in the TARGATT™ High resolution Protein Library Screening.

TARGATT™ Library Screening Overview: The TARGATT™ protein evolution screening system supports a simple and efficient workflow in the well-researched HEK293 cell line (figure 1). TARGATT™-HEK (H11) master cell line is engineered with a “attP” integrase recognition “docking site” in the human HIPP11 (H11) safe harbor locus.

  1. Make the plasmid library for the gene variants into the TARGATT™ donor plasmid containing the “attB” integrase recognition sequence.

  2. Make the knock-in HEK293 cell library, containing only a single copy of each variant per cell.  

  3. Use a cell-based selection assay to enrich your variants.

  4. The isolated cells can be subjected to further screening or the desired variant can be used for phenotype analysis or testing.

 

TARGATT™ Technology Applications:

  • SITE-SPECIFIC INTEGRATION OF TRANSGENES (Patent Pending)
  • NOVEL INTEGRATION SITES AND USES (Patent Pending)

__________________________________________________________

TARGATT™ Screen Master Cell Lines
The TARGATT™ Screen Master Cell Lines were engineered using a split-cassette selection/screen system. This allows us to obtain clean results with little background. We separated the promoter and the transgene. The promoter was inserted in the chromosome at a safe-harbor locus, and the transgene is carried by the donor plasmid. This system only allows expression of the insert if there is a site-specific gene insertion at the safe-harbor locus that contains the promoter. If random integration were to occur, the gene would not have a promoter and therefore would not be expressed.

Applied StemCell integrated this system into HEK293 and CHO cells. Both cell lines have reported high integration efficiencies and medium to high levels of protein expression.

Figure 2: Schematic of the split-cassette selection/screen system.

Publications

TARGATT™ Master Cell Lines

  • 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 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., 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?

An Applied StemCell technical expert is happy to help, contact us today!

Google