• TARGATT™ Site-Specific Knock-in Cell Line Service

TARGATT™ Site-Specific Knock-in Cell Line Service

Applied StemCell’s proprietary site-specific TARGATT™ technology can be used to generate stable, knock-in of large transgenes in cell lines, including stem cells, very efficiently and quickly.  Knock-in is mediated by PhiC31 integrase at a pre-engineered “docking site” in an intergenic, transcriptionally active genomic locus (safe harbor locus) for guaranteed gene expression without disruption of internal genes. This technology allows only a single-copy integration with very high efficiency with or without clonal selection.

Use our TARGATT™ technology to generate your ”Master” cell lines, reporter/tag lines, for iPSC generation, conditional gene expression models and more.

Also, try our ready-to-use TARGATT™ Master Cell Lines to knock-in transgenes in your own lab.

Products and Services

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Support Materials
Application Notes

Combining CRISPR & TARGATT™ in hiPSCs to Enable Large DNA Insertion 

1. CRISPR: Insertion of 70 bp attP docking site to create TARGATT™ Master Cell Line

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Figure 1. Strategy to knock-in attP "docking sites" into hiPSC Rosa26 locus to generate a TARGATT™ iPSC Master Cell Line

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Figure 2. Clone 3 shows heterozygous insertion of attP sequence.

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Figure 3. Sequencing of clone C3 shows insertion of 70 bp attP docking site in Rosa26 locus (red) 

 2. TARGATT™: Large DNA Insertion in iPSC Master Cell Line

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Figure 4. Schematic illustration of the integrase based knock-in in hiPS cells

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Figure 5. PCR Gel electrophoresis to confirm insertion of 5.6 kb fragment in TARGATT™ iPS Master cell line.

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Figure 6. PCR to confirm gene knock-in into Rosa26 locus in TARGATT™ Master iPSC line.

Site-Specific Knock-in of TARGATT™ sites

mCherry red image - Knock-in Cell Line- human iPSC line
Figure: Using mCherry reporter (left) in human iPSC line. Original Cell Line: hiPSC (right).
FAQs
I am interested in making a master TARGATT™ cell line using my own cell line as a parental cell line. Can you take my cancer cell line, for example, to generate a master cell line using your service?
What is the Gene Knock-in Technology used to generate master cell lines?
How long does it take to make a master cell line?
Is there a size limit on DNA to be inserted into the genome (to attP site)?
What is the final deliverable product?
What safe harbor loci are available to place the TARGATT™ docking site? Can you recommend one for my cell lines?
I got my master cell line. Do you provide plasmid so that we can construct Knock-in vector with our gene of interest?
Do you have off-the-shelf TARGATT™ Master cell lines?
Technical Details

Applied StemCell’s proprietary, TARGATT™ that allows for the site-specific integration of large DNA fragments, more efficiently and faster. The novel TARGATT™ technology uses the PhiC31 bacteriophage integrase to mediate an irreversible recombination between a pre-engineered “attP” docking-site and an attP recognition-sequence on the donor vector “attB”, for stable gene integration. The attP docking sites can be engineered into a specific or intergenic, transcriptionally active genomic locus (safe harbor locus), using CRISPR/Cas9. The transgene integration is site-specific, stable and with guaranteed expression.

Applications:

  • Precise comparison of different genes from more than one cell line
  • Generate a master cell line expressing different reporter genes
  • Gene overexpression
  • Inducible gene expression models

Applied StemCell is now providing a TARGATT master knock-in cell line generation service

  • You send us your favorite cell lines
  • We will insert the attP docking sites at specific loci
  • Fast: get your transgenic cell line in 3 months!
Publications

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 Research42(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 America108(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 Sciences108(19), 7659-7660.

Tet inducible mice generated by TARGATT™

Advantage of Hipp11 (H11) locus

Applications for mice generated by TARGATT™ (and cited/published articles

  • 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 researchhttps://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 Reports7, 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. Development144(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 Cell31(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 & Medicine99, 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 Cell60(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. eLife3, 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 Biology426(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 bulletin59(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 Sciences17(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 Genetics46(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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