TARGATT™ Site-Specific Knock-in Cell Line Service


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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.

The TARGATT™ technology can be used to generate stable, knock-in cell lines, including stem cells, very efficiently and quickly. TARGATT™ provides transgene expression and a knock-in cell line in one step.

Generation of TARGATT™ master knock-in cell line with attP docking sites allows:

  • Site-specific integration at pre-defined loci.
  • High integration efficiency with no disruption of endogenous genes
  • Single-copy integration and stable expression
  • Single-step transfection to create stable cell lines
  • No massive clone screening required
  • Inclusion of large transgenes up to 22 kb

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!
Need Site-Specific Knock-in of iPSCs? Applied StemCell can do that too!
 
Also,  Check out our ready to use TARGATT™ Master Cell Lines to knock-in transgenes in your own lab:
TARGATT™-CHO Master Cell Line (hH11 locus) - Inquire

Site-Specific Knock-in of TARGATT™ sites

mCherry red image - Knock-in Cell Line- human iPSC line Original cell line image - Knock-in Cell Line- hiPSC
Figure: Using mCherry reporter (left) in human iPSC line. Original Cell Line: hiPSC (right).

Technical Details

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.

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

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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.

FAQ

FAQ for Gene Knock-in Technology and TARGATT™ Master Knock-in Cell Line Service

1. 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?

Yes.

2. What is the Gene Knock-in Technology used to generate master cell lines?

We use CRISPR/Cas9 to generate master cell lines by inserting TARGATT™ attP sites at a desired locus.

3. How long does it take to make a master cell line?

3-5 months, depending on the nature of the cell line.

4.  Is there a size limit on DNA to be inserted into the genome (to attP site)?

So far we have successfully inserted a transgene of 20kb using this method.

5. What is the final deliverable product?

We ship at least 2 vials, each at 0.5x10^6 cells/vial, cryopreserved cells per clone with a report of the project. Additional clone(s) and vial(s) are available upon request.  For fee-for service projects, you can also have the CRISPR and TARGATT™ vectors upon request.

6. What safe harbor loci are available to place the TARGATT™ docking site? Can you recommend one for my cell lines?

We have several ready-to-use safe harbor loci to choose from. They are Rosa26, H11, and AAVS1. Docking site insertion can be customized to other loci base on your project.

7. I got my master cell line. Do you provide plasmid so that we can construct Knock-in vector with our gene of interest?

Yes.

8. Do you have off-the-shelf TARGATT™ Master cell lines?

Currently, we have human iPSC and mouse C57/BL6 ESC and iPSC lines. CHO cells will be launched soon. We are happy to discuss for other cell lines as Custom Cell Line Services.

 

References

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)

  • 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.
  • Matharu, N., Rattanasopha, S., Maliskova, L., Wang, Y., Hardin, A., Vaisse, C., & Ahituv, N. (2017). Promoter or Enhancer Activation by CRISPRa Rescues Haploinsufficiency Caused Obesity. bioRxiv, 140426.
  • 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 investigation126(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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