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!
- 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 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.
AST-1400 and AST-1405 TARGATT™ CHO-K1 Master Cell Line & Knockin Kit contain the “attP” docking-site at two different safe-harbor locus, H11 or ASC2 respectively. These two kits are suitable for research applications involving gene overexpression and high-level expression of recombinant proteins and other biologics in a rapidly expanding bioproduction industry and for other applications
The AST-1409 and AST-1410 TARGATT™ CHO-K1 Kits (H11) for antibody library screening were designed for library construction. The CHO cell line contains the “attP” docking-site at the H11 safe-harbor locus. The AST-1409 kit is for scientists who would like to use FACS, and the AST-1410 kit is ideal for drug selection.
Yes, that is available. What we can provide is the Master Cell Line and the vector in which you can build your library in.
For Library Construction:
AST-1306: TARGATT™ HEK293 Kit (H11) for Antibody Library Screening (Drug Selection); is ideal for drug selection
AST-1307: TARGATT™ HEK293 Kit (H11) for Antibody Library Screening (FACS); ideal for fluorescence-activated cell sorting (FACS)
For Knockin Only:
AST-1305: TARGATT™ HEK293 Master Cell Line & Knockin Kit
- 15 μg of the integrase included in the kits allows for approximately 27 transfections (if 0.5 μg of integrase is used per 24wp well).
- Scale-Up (5X DNA Needed): 15 μg of the integrase included in the kits allows ~5 transfections (if 2.5 μg per well is added to 6-well plates)
Yes, additional integrase can be purchased. Please contact ASC for more information.
Contact us today to set up a free consultation and learn more about how we can work with you to drive your research forward!
Yes, contact us today to set up a free consultation with one of our experts!
Yes, the TARGATT™ system can be integrated into the cell line of your choice.
Following the purchase of the kit, you will receive all plasmid vector maps and the necessary sequence data to carry out your project. The product datasheet includes an example of how to design the cloning/library primers. If you have any questions about how to design your primers please contact Applied StemCell.
No, no promoter is included in the cloning plasmids. To learn more contact ASC today.
Outgrowth depends on library cloning efficiency, and how well E.coli handle the plasmids. Generally, we use 10 uL of the 1 hour outgrowth to make several serial 10-fold dilutions, and plate those to estimate library size/coverage.
The remaining outgrowth (~950-990 uL) is used to seed at least 140 mL of Terrific Broth with kanamycin. We will use more Terrific Broth if we expect the efficiency to be higher. We shake at 300 RPM in baffled 250 mL flasks for 18 hours, then midi-prep with the Macherey Nagel kit.
To figure out if these conditions are adequate, you can monitor the 140 mL growth (e.g. check OD600) at several time points, e.g. 8-18 hrs. If the cells are no longer growing, then you should be pelleted and frozen. Alternatively, if you continue to grow after 18 hours, then the incubation could be extended to 24 hours so that yield is improved.
If you have any other questions, please contact us.
The system in the paper uses inserted phiC integrase. By adding exogenous CMV-integrase plasmid, we see an improved efficiency.
TARGATT™ HEK293 library kit is better suited for large library screening.
Neon gave us the best results so far.
Pricing will depend on the size of the library. Please give us a size and we will get a pricing for you.
TARGATT™ has a higher integration efficiency. For libraries, this is important because a higher efficiency means that larger variant pools can be established, which increases the odds and power of success. E.g. you will be more likely to find antibodies in your library that bind as desired, and you will also be more likely to find strong binders.
With the system used in our bioproduction lines (AST-1200, 1400, 1405), every donor plasmid has at least one promoter that drives expression of the downstream protein(s). This is fine for production because every plasmid is supposed to be identical (i.e. want same protein or proteins expressed in every cell).
For library screening, this is not desirable, because we need a one-to-one genotype-phenotype connection. E.g. when a cell is isolated because it expressed an antibody (on its surface) that binds the desired antigen, we don't want to have to do detective work (further screening of 10s to 100s of variants) to figure out which antibody sequence is responsible. We want that cell to have been expressing a single antibody, and we only want to see one clear result when performing sequencing (of the amplified PCR product).
The bioproduction cells would be best. We say this because the bioproduction CHO cells are more optimal for secreting antibodies, and it won't make a difference for your application if cells are making a variety of different variants.
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*Featured in Informa Connect's eBook: Antibody Discovery, Selection & Screening.
Poster and Poster Presentation:
Applications for High Resolution Protein Screening:
- Directed evolution (vaccine development, drug screening, cell-based gene therapy)
- Genome-wide screening
- Bioproduction/ stable cell line generation
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.
Make the plasmid library for the gene variants into the TARGATT™ donor plasmid containing the “attB” integrase recognition sequence.
Make the knock-in HEK293 cell library, containing only a single copy of each variant per cell.
Use a cell-based selection assay to enrich your variants.
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.
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 ONE, 14(7), e0219842.
Transgenic Mouse Book Chapters
- Chen-Tsai, R. Y. (2020). Integrase-Mediated Targeted Transgenics Through Pronuclear Microinjection. In Transgenic Mouse (pp. 35-46). Humana, New York, NY.
- Chen-Tsai, R. Y. (2019). Using TARGATT™ Technology to Generate Site-Specific Transgenic Mice. In Microinjection (pp. 71-86). Humana Press, New York, NY.
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 Research, 42(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 America, 108(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 Sciences, 108(19), 7659-7660.
Tet inducible mice generated by TARGATT™
- Fan, X., Petitt, M., Gamboa, M., Huang, M., Dhal, S., Druzin, M. L., … Nayak, N. R. (2012). Transient, Inducible, Placenta-Specific Gene Expression in Mice. Endocrinology, 153(11), 5637–5644. http://doi.org/10.1210/en.2012-1556.
Advantage of Hipp11 (H11) locus
- Hippenmeyer, S., Youn, Y. H., Moon, H. M., Miyamichi, K., Zong, H., Wynshaw-Boris, A., & Luo, L. (2010). Genetic Mosaic Dissection of Lis1 and Ndel1 in Neuronal Migration. Neuron, 68(4), 695–709. http://doi.org/10.1016/j.neuron.2010.09.027.
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. eLife, 8.
- 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 one, 14(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 research. https://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 Reports, 7, 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. Development, 144(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 Cell, 31(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 & Medicine, 99, 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 Cell, 60(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. eLife, 3, 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 Biology, 426(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 bulletin, 59(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 Sciences, 17(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 Genetics, 46(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)