TARGATT™ CHO and HEK293 Cells For Antibody Library Construction
TARGATT™ technology enables faster and more 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.
At ASC, we integrated TARGATT™ site-specific knock-in 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.
The TARGATT™ master cell lines are ideal for antibody library construction. Our experts have successfully completed several custom TARGATT™ HEK293- and CHO-based library construction projects which have enabled the optimization of our off-the-shelf TARGATT™ HEK293 and CHO library screening kits (Drug selection and FACS kits available). These high-quality kits allow you to build your library in your own lab! Explore our products below or contact us today to schedule your free consultation.
ASC 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
- Fully customizable library construction service
- Four ready-to-use TARGATT™ antibody library screening kits for you to build your own library
Licensing and Evaluation Programs Available!
Products and Services
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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™ 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™ 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.
eBook: TARGATT™ Technology For Antibody Discovery and Screening (March 2021)
*Featured in Informa Connect's eBook: Antibody Discovery, Selection & Screening.
*Featured in Informa Connect's eBook: Antibody Discovery, Selection & Screening.
Poster and Poster Presentation:
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)
Can we in this case fuse the signal sequence directly to the C-terminus of the P2A tag or do we need to attach a linker sequence?
Does the vector cassette (mCHERRY-P2A-LacZ) has PaqC1 cleavage sites (CACCTGC)?
You can also request the construction of a library, but can you give us an estimate of the price?
Pricing will depend on the size of the library. Please give us a size and we will get a pricing for you.
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?
TARGATT™ HEK293 library kit is better suited for large library screening.
Which TARGATT™ CHO Kit should we use if we are not isolating single antibodies (eg. the best binders)?
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.
What is the difference between AST-1400, AST-1405, AST-1409, and AST-1410?
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.
Which TARGATT™ CHO Kit should I use?
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).
What is the difference between the AST-1306/AST-1307 antibody screening kits and the AST-1305 knockin kit?
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
Why should I use ASC's HEK293/CHO TARGATT system rather than phage-display or other display methods?
It allows for mammalian antibodies with correct post-translational modifications. There is no variation from one clone to another (single copy and site-specific insertion). The same library diversity and coverage as phage display can be achieved, and single-copy expression is >150mg/L. Also, phage/yeast display does not allow for use of full antibodies. Researchers express and screen fragment libraries using microbes, then hope that they can find a way to preserve the desired binding when the fragment is placed in a full antibody.
Can I use this system in my own cell line?
Yes, the TARGATT™ system can be integrated into the cell line of your choice.
Can you tell us a bit more about the screening process, does it require clonal selection?
If used for the CHO stable cell line you have a choice. You can make clones so that you have a stable cell line. Sometimes people want to conduct quick experiments to look at expression and, in that case, you would not have to do a single clone, you can just look at a pool. Our system allows you to enrich the gene insertion up to 98% in CHO cells. The pool will pretty much do the work. In that case, you would not need to do cloning.
Could you describe the business model to access your technology?
1) We can provide service; you tell us what your goal is and we will provide a fee for service. 2) We can provide a nonexclusive license. In terms of licensing, there are two options: a) Research use only b) Commercial License (case by case basis)
You can do up to 10e9, but is it possible to provide a protocol for building a large-scale library?
Yes. There are also many commercial companies that offer library building services. We often outsource the library building part to our partner companies.
Is the library constructing plasmid available for us to make our own library by ourselves?
Yes, that is available. What we can provide is the Master Cell Line and the vector in which you can build your library in.
What can the TARGATT™ library system be used for?
Our TARGATT™ library system can generate stable cell line libraries for mammalian cell display-mediated antibody engineering, protein evolution screening, mammalian two-hybrid (M2H) screens, and more.
Why is TARGATT™ better?
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.
How long does it take to obtain a pool? Could this be used for high-throughput Ab production?
To get a pool it only takes a couple of weeks. Yes, we have developed a high-throughput format in plates.
AST-1307: Is there a marker for detection of integration efficiency?
mCherry is used for the detection of integration efficiency (non-integrated donor will not fluoresce).
AST-1306 & AST-1307: Is additional integrase available?
Yes, additional integrase can be purchased. Please contact ASC for more information.
AST-1306 & AST-1307: How many screenings can we do with these kits?
- 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)
AST-1306 & AST-1307: I want to test this. What is the next step?
Contact us today to set up a free consultation and learn more about how we can work with you to drive your research forward!
With more copies will I have a higher yield?
For bioproduction, multiple copies does sometimes mean more yield, but it takes a lot of work to screen clones that (i) have higher yield and (ii) maintain that yield (i.e. do not get silenced). Screening clones (Western, sequencing, etc.) for one or a few antibodies might be reasonable, but it's not if it has to be done for multiple antibodies at once (5, 10, etc.).
AST-1306 & AST-1307: Are you able to make a construct library for the GOI that I have?
Yes, contact us today to set up a free consultation with one of our experts!
Why do we need to use Golden Gate Assembly for the cloning?
There are some restriction enzyme sites in the donor plasmid which you can use for cloning.
AST-1409 and 1410: I am interested in the kit, what information can I obtain in order to insert my GOI?
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.
AST-3080 and 3081: Do you disclose the termination site?
AST-3080 and 3081: Do you disclose the promoter, and its sequence?
No, no promoter is included in the cloning plasmids. To learn more contact ASC today.
Why do we need to use these special 10G SUPREME SOLO cells for transformation?
All the reagents listed in the protocol are recommended based on our in-house testing. You definitely can use a similar/alternate reagent. We recommend that you may perform a small-batch test using your preferred reagents.
AST-1409: A plasmid was transfected in by electroporation or lipofectamine?
AST-1409: A plasmid was introduced by lentiviral or other retrovirus methods?
None of the above
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
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.
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?
The system described on the website is an improved version of the system in the paper.
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?
The system in the paper uses inserted phiC integrase. By adding exogenous CMV-integrase plasmid, we see an improved efficiency.
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.
Antibodies can be expressed on the cell surface by adding transmembrane domains. Hch/Lch can be expressed in the form of (1) two separate expression cassette; or (2) under one expression cassette separated by 2A sequences.
Information on gene transfer in CHO is shown in Neon, but is there any information in other devices (NEPA, Nucleofactor2b, etc.)?
Neon gave us the best results so far.
We recovered the AST-1408-1 cells sent with the AST-1409 kit using a different media (CD FortiCHO™), and the cells did not recover well (low viability). Could I please get some guidance on this issue? I think the issue was the seeding density.
We recommend using BalanCD CHO Growth A medium with L-Glutamine as a culture medium.
Note: CD FortiCHO™ medium is used to support mainly protein expression in suspension batch culture, so we would not recommend CD FortiCHO™ medium for cell culture.
Once cells are thawed, re-suspend the cell pellet in pre-warmed fresh complete culture medium at a density of 2.5 – 3.0 x10^5 viable cells/mL, in a fresh 125 mL Erlenmeyer shaker flask. Since there are 1 million cells in the vial, the cells should be resuspended to ~4 mL and placed in a 125 flask (https://htslabs.com/og/). Place the flask on an orbital shaker platform rotating at 130rpm, in a 37°C and 5-8% CO2 incubator; this is really important for suspension cell growth. After three days, if cell viability is above 5*10^5/ml, add some complete culture medium to adjust cell density to 3.0x10^5 viable cells/mL, but don’t take any cell out until cell viability is above 5*10^6/ml. Once cell viability is above 5*10^6/ml, cells are ready to be passaged.