TARGATT™ HEK293 Master Cell Line for Site-Specific Knockin
Mammalian Cell-based Protein/ Antibody Discovery & Screening! The TARGATT™ HEK293 Master Cell Line provides an efficient way to generate site-specific, stable, knockin cell lines (even large transgenes) and build large mammalian cell libraries:
- High knockin efficiency: with enrichment (up to 90%) or without (up to 40%)
- Site-specific: H11 locus; active, genomic hotspot
- Single copy integration
- Uniform, high level gene expression
- Unidirectional integration
- Overcomes challenges posed by random integration
The TARGATT™ HEK293 Master Cell Line is ideal for drug screening, toxicity studies, CNS disease or cell modeling, and regenerative medicine studies.
Yes, the TARGATT™ 24 CMV-MCS cloning plasmid (AST-3064) contains mCherry and will express mCherry after recombination.
We cannot disclose the growth and purification protocols of the AST-3065 and AST-3201 plasmids for proprietary reasons. The amount of the plasmids provided in the kit is sufficient for 9 transfections (3 triplicate transfections in a 24-well plate, according to the provided protocol). Additional plasmids (AST-3065 and AST-3201; $550/15 µg each plasmid) are available for those who have previously purchased the AST-1305 TARGATT™ HEK293 Master Cell Line and Knockin Kit.
No. The TARGATT™ 24 (CMV-MCS-attB) plasmid is meant only for single transgene integration to generate stable knockin 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 (with mCherry for FACS enrichment/ 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).
In addition to the TARGATT™ HEK293 Master Cell Line and Knockin Kit, Applied StemCell also provides TARGATT™ master cell lines in the 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 knockin 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.
No. the plasmids AST-3050 and AST-3051 cannot be used with the TARGATT™ HEK293 Master Cell Line. If you are interested in a custom plasmid other than the ones provided in the kit, please inquire.
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 TARGATT™ HEK293 Master Cell Line (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.
Yes, we can generate a TARGATT™ Master Cell Line in your cell line of interest.
Yes, we can generate a TARGATT™ Master Cell Line with a docking site at your locus of choice. We have currently master cell lines in HEK293, CHO, and hiPSC backgrounds, with docking site in the proprietary H11 and ASC2 safe harbor loci.
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.
Yes, diluting in 10 μg/mL blas medium does work.
*Featured in Informa Connect's eBook: Antibody
Discovery, Selection & Screening.
Schematic Representation of the Transgene Integration in the TARGATT™ Master Cell Line
Figure 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
Figure 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
Figure 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.
Comparing TARGATT™ and existing gene editing technologies for generating stable knockin cell lines:
Please note that the TARGATT™ integrase-based knockin 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 knockin efficiently. We have master cell lines in HEK293, CHO and hiPSC background. We can also custom engineer a landing pad and generate a Master Cell Line in the cell line of your choice. Please inquire for further details.
TARGATT™ HEK293 Master Cell Line and Knockin Kit - A valuable research tool to generate stable knockin cell lines and large, isogenic, mammalian cell libraries very efficiently!
The TARGATT™ Knockin 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 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 stably integrated knockin cell lines.
Of note, the landing pad in the TARGATT™ HEK293 master cell line is engineered into the well-defined, transcriptionally active, intergenic H11 locus (safe harbor locus/ genomic hotspot). This locus enables high level expression of the integrated gene-of-interest without disruption of internal genes and gene silencing commonly seen with random integration.
Advantages of the TARGATT™ HEK293 Master Cell Line:
- High efficiency and stringent gene knockin
- Site-specific integration into the H11 genomic hotspot well-defined safe harbor locus
- Single gene knockin: one variant - one locus - one cell line
- Unidirectional integration for stable knockin cell lines
- 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™ HEK293 Master Cell Lines and Knockin Kit 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 knockin into cell lines and for library generation.
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
How is the TARGATT™ HEK293 Master Cell Line different from other technologies for purpose of site-specific integration of transgenes and stable cell line generation?
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.
How does the TARGATT™ HEK293 Master Cell Lines compare to other HEK293 master cell lines and kits using similar site-specific technologies?
- 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 knockin 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.
How does the TARGATT™ technology compare to nuclease-based (Cas9) and other systems such as Flp, Cre, etc.?
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 knockin 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 can 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 Application Notes section for more comparisons between the TARGATT™ and other commonly used technologies.
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
- 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.
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- 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.
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