• 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

Viewing 1-5 of 5 products
per page
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

technical-targatt-ipsc-crispr-attpKI-1

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

technical-targatt-iPSC-clone-with-attp-2

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

technical-targatt-ipsc-seq-align-attp-3

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

technical-targatt-ipsc-schematic-targatt-ki-4

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

 technical-targatt-ipsc-pcr-to-confirm-KI-5

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

technical-targatt-ipsc-pcr-to-confirm-ki-Rosa26locus-6

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

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 ONE14(7), e0219842.

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)

 

Have Questions?

An Applied StemCell technical expert is happy to help, contact us today!