TARGATT™ Knock-in Master Cell Line and Knock-in Kit (HEK293, CHO, and iPSC)
The TARGATT™ Master Cell Lines (HEK293, CHO, and iPSC) and transgene knock-in kits are an efficient way to generate stable, isogenic, knock-in cell lines and build large mammalian cell libraries, even large transgenes.
- High knock-in efficiency: with enrichment (up to 90%) or without (up to 40%)
- Site-specific: well-defined, active, intergenic safe harbor locus
- Single copy integration
- Uniform, high level gene expression
- Unidirectional integration
- Overcomes challenges posed by random integration
The TARGATT™ Master Cell Lines are ideal for research in immunocompatible cell therapy & directed-differentiation (iPSCs); gene regulatory elements, protein evolution, antibody/ recombinant protein expression and high-yield bioproduction studies (HEK293 & CHO cells).
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ASC's Custom Services for Cell Line and Animal Model Generation, and Downstream Assays for Drug/ Toxicity Testing
No. The TARGATT™ 24 (CMV-MCS-attB) plasmid is meant only for single transgene integration in order to generate stable knock-in cell lines. It cannot be used for mammalian library construction as it would lead to expression of many variants from non-integrated plasmids for an extended and undetermined period of time, and would require diluting out the plasmids.
For library construction, we have two TARGATT™ library cloning plasmids (one has mCherry for FACS enrichment, the other has blasticidin resistance for drug enrichment) that can be used with our TARGATT™ HEK293 Master cell line to efficiently build mammalian cell libraries. Please inquire for details.
The datasheet (product manual) provides sequence for universal primer sets (upstream primers, downstream primers) to confirm site-specific insertion of the gene of interest (GOI) in the appendix section. The datasheet also includes sequence for a human control primer which we recommend you use to check the integrity of the cells and genomic DNA (gDNA).
No. We can only share the plasmid map for the plasmids provided in the kit. We will not be able to share the sequences for proprietary reasons.
No. the plasmids AST-3050 and AST-3051 are not for use with the master cell lines. If you are interested in a custom plasmid other than the ones provided in the kit, please inquire.
The TARGATT™ integrase system is designed based on serine integrases which catalyze irreversible site-specific transgene insertion via recombination between two distinct integrase-recognition/ attachment sites, attP and attB. The difference of the TARGATT™ HEK293 Master Cell Line is its high efficiency in gene insertion (up to 45% compared to 5% using other systems) and its specificity with very low off-target integration profiles.
Another advantage of the TARGATT™ HEK293 Master Cell Line is its fast Blasticidin enrichment and mCherry selection (2-3 days vs weeks) that allows you to purify the gene edited cells most effectively.
No, you cannot use hygromycin or G418 in your cell culture system.
No. The integration of the plasmid backbone should not affect the expression of your gene of interest in the HEK293 cell lines (internal test with hundreds of transgenes). The TARGATT™ 24 Cloning Plasmid has a proprietary design and the backbone has been engineered with regulatory elements to ensure site-specific integration into a transcriptionally active safe harbor locus and reduce random integration and gene silencing.
In addition to the TARGATT™ HEK293 Master Cell Line and knock-in kit, Applied StemCell also provides TARGATT™ master cell lines in Chinese Hamster Ovary (CHO) cell and induced pluripotent stem cell (iPSC) backgrounds. The different cell line backgrounds enable the use of TARGATT™ site-specific integration technology to efficiently generate stable knock-in cell lines for a multitude of purposes such as protein screening, bioproduction, cellular differentiation and directed-differentiation of cell lineages, immuno-compatible cell line generation, and more.
We also offer custom TARGATT™ Master Cell Line Generation service where we can engineer the TARGATT™ landing pad into any cell background and locus of your choice, and design the corresponding plasmids.
We have successfully integrated plasmids up to 8 kb with more than 40% efficiency and without enrichment/ selection (considerably higher than similarly available technologies), in the current system. 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. With selection/ enrichment, the size of the integrated plasmid should not be affected by efficiency.
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.
- High basal integration efficiency (up to 40%) even without selection or enrichment. With selection, modified cells can be enriched up to or higher than 90%.
- Unidirectional recombination ensures stable knock-in cell lines
- Single-step transfection procedure – no need for re-targeting
- Stringent and low off-target profile for random integration
- Integration locus, H11 is well-defined, intergenic, and transcriptionally active safe-harbor locus
- The H11 locus expresses high level of protein, uniformly and consistently
- Enables the generation of isogenic cell lines efficiently
- With selection/enrichment, stable-line pools can be obtained within 2 weeks.
The TARGATT™ technology is based on serine integrases which catalyze efficient site-specific DNA cleavage, DNA strand exchange and ligation without help from outside proteins. This permits knock-in efficiencies superior to those that are possible with nucleases like Cas9, which leave cleaved DNA to be dealt with by the host repair machinery.
Bi-directional recombinases such as Flp and Cre also permit avoidance of the cell DNA repair machinery, but are highly inefficient for net-integration due to the extreme kinetic favorability of the excision reaction. I.e., while they are able to mediate the initial genomic-recombination event with the same efficiency, they then proceed to catalyze the excision reaction with high efficiency (as the substrates are now physically linked). Serine integrase attL and attR complexes do not synapse, so this subsequent excision is blocked, and thus a high net-integration efficiency can be achieved.
You can also review the table in the Technical Details section for more comparisons between the TARGATT™ and other commonly used technologies.
Now Launching! TARGATT™ HEK293 Master Cell Line and Knock-in Kit - A valuable research tool to generate large, "ISOGENIC" mammalian cell libraries very efficiently!
The TARGATT™ Knock-in Master Cell Line and Kit uses integrase-based integration of a transgene into a preselected intergenic and transcriptionally active genomic locus (hROSA26, hH11, hAAVS1 or other safe harbor loci) engineered with an integrase recognition attP docking site or “landing-pad). Applied StemCell provides landing-pad ready TARGATT™ Master Cell Lines and Kits in three cell line backgrounds: HEK293, CHO, and hiPSC.
The knock-in kit provided with the master cell line includes a TARGATT™ cloning plasmid is used to generate the donor plasmid containing the gene of interest (transgene) and also contains a corresponding integrase-recognition attB sequence. When the donor plasmid is transfected into the cell line, the integrase catalyzes the integration of the transgene at the attP-attB sites. This integration is unidirectional which results in a stably integrated knock-in cell line.
The landing-pad in the master cell line is engineered into a well-defined, transcriptionally active, intergenic locus (safe harbor locus) that enables high level expression of the integrated gene-of-interest, and without disruption of internal genes and gene silencing commonly seen with random integration.
These TARGATT™ technology and master cell lines can be utilized for generating stable and isogenic, knock-in cell lines and libraries for a variety of applications including reporter gene expression, gene knockdown, conditional/ inducible gene expression, gene overexpression, directed-differentiation of iPSCs, inserting an overexpression cassette for generating immune-privileged iPSCs, antibody expression libraries, pilot bioproduction of recombinant protein, and disease modeling.
Advantages of TARGATT™ technology:
- High efficiency and stringent gene knock-in
- Site-specific integration into a well-defined safe harbor locus
- Isogenic knock-in cell lines: one variant - one locus - one cell line
- Stable gene integration with unidirectional integration
- Fewer cell counts and library sizes needed required
- Permits non-viral high-throughput library screens
- Overcomes challenges such as random insertion, gene silencing, multiple copy gene integration, ablated gene expression.
The TARGATT™ Master Cell Lines and Knock-in Kits combines the scalability, affordability, and ease-of-use of bacterial/ yeast systems and the advantages of using mammalian cells (closer to human environment and post-translational modifications) for efficient and stable gene knock-in into cell lines and for library generation.
Comparing TARGATT™ and existing gene editing technologies for generating stable knock-in cell lines:
Please note that the TARGATT™ integrase-based knock-in technology requires the master cell line to have a landing pad (docking site) engineered into the cell at the chosen locus to be able to knock-in efficiently.
If you would like, Applied StemCell provides custom service to engineer a landing pad and generate a Master Cell Line in the cell line of your choice. Please inquire for further details.
Potential Applications include but are not limited to:
Stem Cell Research
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
- 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 TARGATT™ technology
- 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)