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Applied StemCell, TARGATT™

HEK293 – high-efficiency library construction/screening

Stable HEK293 for AAV production

Fast turnaround

Library Construction & Screening

With our proprietary TARGATT™ technology for site-specific integration of large DNA fragments, we’ve developed a series of TARGATT™ “master” cell lines, including engineered HEK293 cells. These cell lines are available as part of our user-friendly TARGATT™ knock-in kits, allowing you to quickly knock in your gene of interest (GOI) in your lab. Our TARGATT™ HEK293 cell lines demonstrate high-level of gene knock-in efficiency and are ideal for library construction and screening in mammalian cells.

TARGATT™ HEK293 Master Cell Line

The TARGATT™ Knockin Master Cell Line and Kit uses integrase-based integration of a transgene into a preselected intergenic and transcriptionally active genomic locus (hH11) pre-engineered with an integrase recognition “attP” docking site or “landing-pad”). The H11 locus is well-defined, transcriptionally active, and located at intergenic region (safe harbor locus/genomic hotspot). This locus enables the high-level expression of the integrated gene-of-interest without disruption of internal genes and gene silencing commonly seen with random integration.

Applied StemCell (ASC) provides landing-pad ready TARGATT™ master cell lines and kits.

The TARGATT™ HEK293 Master Cell Line and Knockin Kit includes a TARGATT™ cloning plasmid that contains an integrase-recognition “attB” sequence and can be used to generate the donor plasmid containing the gene of interest (transgene). When the donor plasmid is transfected into the master cell line along with the integrase expression plasmid (also provided in the kit), the integrase catalyzes the integration of the transgene at the attP-attB sites. This integration is unidirectional which results in a stable integrated knock-in cell line.

Main applications
a. Library construction and screening

The TARGATT™ HEK293 Master Cell Lines and Knockin Kit combines the scalability, affordability, and ease of use of bacterial/yeast systems for library screening. Given its high integration efficiency, the TARGATT™ HEK293 system provides up to 107 to 109 library coverage (similar to bacterial/yeast systems), offering the highest library coverage in mammalian cells on the market.  The advantages of using mammalian cells are proper post-translational modifications and other epigenetic modifications that are missing in bacterial or yeast cells. 

The TARGATT™ Screening System:
Project TARGATT™ Library Sizes

Advantages of TARGATT™ System

Advantages of TARGATT™ System

b. Generate stable TARGATT™ HEK293 for AAV production. The current AAV production uses tri-plasmid co-transfection to transiently express AAV components in HEK293 cells. The yield is very low and costly. Using TARGATT™, we can generate stable HEK293 cells with the cap-rep components inserted in the genome, and the gene of interest will be inserted at a safe harbor genomic locus. We are currently looking for partners to make HEK293 cells for AAV production.

TARGATT™ HEK293 Master Cell Lines:

  • Single-Copy Knock-In: Achieve precise integration with one cell, one docking site, and one inserted transgene.
  • Site-Specific Integration: Target high-expression, safe harbor loci (H11) for optimal results.
  • High Efficiency: Enjoy integration rates of over 40% without selection and over 90% with drug selection.
  • Cost-Effective: Save time and resources by eliminating the need for virus packaging compared to lentiviral library screening.
  • BSL1 Compatible: Suitable for use in BSL1 laboratory environments.

TARGATT™ HEK293 Library Kits:

  • Construct large cell libraries efficiently
  • Develop a mammalian display system with enhanced efficiency
  • Achieve mammalian display system library sizes comparable to those of E. coli and yeast

Get Started with Applied StemCell

Contact Information
Reach out to us directly through our contact page for any inquiries. Our team is ready to assist you with your TARGATT™ HEK293 cell needs.

Request a Quote
Requesting a quote is easy. Provide us with the details of your project, and we will deliver a comprehensive and competitive quote tailored to your specific requirements.

  • Application Notes

    Potential Applications include but are not limited to:

    Immuno-oncology

    • CAR affinity/efficiency
    • CAR specificity and safety screening
    • “Universal” CAR-T cell
    • Discover novel immune targets, checkpoints

     

    Antibody Discovery

    • scFv screening
    • Off-target screening with membrane protein library
    • Bioprocessing/ bioproduction

    Protein evolution

    • Enzyme activity and specificity (Cas9, DNA modification enzymes)
    • AAV capsid specificity and efficiency
    • Screening for regulatory elements (promoters, splicing regulators), post-transcriptional regulation
    • Receptor identification: Ion Channels; GPCR

    Stem Cell Research

    • Directed-differentiation to cell-lineages
    • Immuno-compatible/ universal iPSC
    • Non-membrane, non-secretory protein library
    • Off-target screening
    • Mammalian two-hybrid assays

  • Case Studies

    Schematic Representation of the Transgene Integration in the TARGATT™ Master Cell Line

    ast-1305-schematicFigure 1. Schematic representation of TARGATT™ site-specific transgene integration mediated by integrase. The TARGATT™-HEK Master Cell Line was engineered with the attP landing pad at the hH11 safe harbor locus. The TARGATT™ plasmid containing the integrase recognition site, attB is used to clone the transgene. The integrase catalyzes an irreversible reaction between the attP site in the genome and attB site in the donor vector, resulting in integration of the gene of interest at the selected H11 locus. The cells containing the gene of interest can be enriched using the selection marker (gray box).

     

     

     

    Confirmation of Site-Specific CMV-MCS Plasmid Integration

    Confirmation of site-specific CMV-MCS plasmid integrationFigure 2. PCR gel electrophoresis to confirm the knockin of TARGATT™ 24 CMV-MCS-attB plasmid mediated by the TARGATT™ Integrase plasmid, after transfection into the TARGATT™ HEK293 Master Cell Line. Two sets of primers were used to confirm knockin: Upstream (512 bp) and Downstream primers (464 bp). The Human control primers (777 bp) was also used as a control to check the integrity of the cells and the genomic DNA (gDNA). Negative control (-) represents cells transfected with the TARGATT™ 24 CMV-MCS-attB plasmid and a mutant TARGATT™ integrase plasmid that is deficient for integration.

    mCherry Expression After Transfection and Blasticidin Enrichment

    mCherry expression 72 hours post-transfectionFigure 3. The mCherry integration into the TARGATT™ HEK293 master cell line. Left: Integration mediated by the integrase 72 hours post-transfection. Cells were transfected with the mCherry positive control plasmid and either the provided TARGATT™ integrase plasmid (+Integrase) or a mutant TARGATT™ integrase plasmid deficient for integration (-Integrase). The mCherry plasmid has no promoter and requires the ubiquitous EF1 promoter in the landing pad after integration to express the reporter gene. The integration efficiency of mCherry knockin into landing pad was >40%, without selection. Right: Blasticidin enrichment of TARGATT™ HEK293 cells with a knocked-in mCherry-blasticidin plasmid. Cell pools (with 20x and 40x split ratio) were enriched in selection medium for 3 weeks (without cell sorting). The enrichment of mCherry was about 90% after blasticidin selection.   Data represents the mean ± SE of two representative experiments done in triplicates.

Frequently Asked Questions

  • What is the safe-harbor H11 Locus?

    The safe harbor locus H11 refers to a specific location on the human genome, located on chromosome 11, which has been identified as a “safe harbor” for inserting exogenous DNA. This means that inserting new genes into this location is less likely to disrupt the normal functioning of the genome, minimizing the potential for unintended effects. The H11 locus on chromosome 11 corresponds to the Complement Factor H (CFH) gene, which is involved in regulating the immune system. The H11 locus has been identified as a safe harbor locus due to its stable expression and lack of association with any known human diseases when used as a site for gene insertion. The H11 locus is often used as a target site for the insertion of therapeutic genes for gene therapy applications, as well as for research purposes in molecular biology and genetics.

  • How did you confirm a single landing pad, was it copy number qPCR?

    Single landing pad insertion was confirmed by site-specific insertion of “colored” plasmid assays as depicted in the following diagram.  Cells that are either green or red confirm a single landing pad (see data figure); whereas cells that are both green and red (overlapping as yellow) have multiple landing pads.

  • Can you share the HEK293 suspension culture protocol? So that we could evaluate whether it is possible for us to make library cells in suspension condition before we commercialize it.

    We highly recommend transfecting your library into our adherent cells because the transfection, knock-in and enrichment efficiencies are much higher.  After the library is enriched, we have had success with gradual adaptation into Cytiva’s serum-free HEK293 media (Hyclone SFM4HEK293 and CDM4HEK293).

  • Does TARGATT™ 24 CMV-MCS cloning plasmid (AST-3064) express mCherry after recombination?

    Yes, the TARGATT™ 24 CMV-MCS cloning plasmid (AST-3064) contains mCherry and will express mCherry after recombination.

  • Could you explain a bit more about why AST-1306 won't express target after transfection? It seems SV40 promoter is already there in donor plasmid.

    The “SV40” sequence present is the 3’UTR/polyA signal sequence.  In addition to this, we have proprietary sequences present in the plasmid backbone that significantly reduce background levels of non-specific transcription.

  • What is the size of the transgene that can be integrated into the TARGATT™ HEK293 Master Cell Line with the specified efficiency?

    We have successfully integrated plasmids up to 8 kb with more than 40% efficiency without enrichment/ selection with the current system (considerably higher than similarly available technologies). With selection/ enrichment, the size of the integrated plasmid should not be affected by efficiency. There is strong literature evidence that supports high efficiency and stringent integration of large transgenes (up to 200 kb) using serine integrases in a variety of cell lines, yeast, drosophila, and animal models.

    Below are a few references:

    • Tasic B, Hippenmeyer S, Wang C, Gamboa M, Zong H, Chen-Tsai Y, Luo L. Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc Natl Acad Sci U S A. 2011 May 10;108(19):7902-7. doi: 10.1073/pnas.1019507108. Epub 2011 Apr 4. PubMed PMID: 21464299
    • Bischof, J., Maeda, R. K., Hediger, M., Karch, F., & Basler, K. (2007). An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proceedings of the National Academy of Sciences of the United States of America, 104(9), 3312–3317. doi:10.1073/pnas.0611511104
    • Venken, K. J., He, Y., Hoskins, R. A., & Bellen, H. J. (2006). P [acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science, 314(5806), 1747-1751.
    • Duportet, X., Wroblewska, L., Guye, P., Li, Y., Eyquem, J., Rieders, J., … & Weiss, R. (2014). A platform for rapid prototyping of synthetic gene networks in mammalian cells. Nucleic acids research, 42(21), 13440-13451.

  • Are the TARGATT™ HEK293 cells adherent?

    Our TARGATT™ HEK293 cells are adherent so you will need to adapt them to suspension. However, we also have TARGATT™ CHO cells, which are suspension cells.

  • After the transgene integration, the attR and attL sequence are left completely or sometimes have indels?

    The attR and attL sites are left completely. I.e. the TARGATT™ enzyme being used forms a tetramer complex that completes the full recombination reaction (four-strand cleavage, 180-degree rotation, ligation) without any help from the host cell’s machinery.

  • If I would like to use a different promoter, what should I do?

    The CMV can be removed by digesting with AscI and an enzyme in the multiple-cloning site (MCS) — e.g. AscI + BsiWI, AscI + HindIII, etc. If you need additional help figuring out how to clone your promoter of interest, please send us the promoter’s sequence. We can reply with advice about how to clone it or propose some other strategy.

  • Publications

    TARGATT™ 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 ONE, 14(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 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™

    Advantage of Hipp11 (H11) locus

    Applications for TARGATT™ technology

    • 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.
    • Chen-Tsai, R. Y. (2019). Using TARGATT™ Technology to Generate Site-Specific Transgenic Mice. In Microinjection (pp. 71-86). Humana Press, New York, NY
    • 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)

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