Skip to main content
SpringerLink
Log in

Using TARGATT™ Technology to Generate Site-Specific Transgenic Mice

  • Protocol
  • First Online:
Microinjection

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1874))

Abstract

The discovery of new gene editing tools in the past several years has moved the transgenic field to a new level. The traditional random transgenesis method by pronuclear microinjection has been largely replaced by targeted or site-specific transgenic technologies without the need of homologous recombination in embryonic stem (ES) cells. In this chapter, I describe detailed protocols of an integrase-based approach, trademarked as “TARGATT™” (target attP), to produce site-specific transgenic mice via pronuclear microinjection, whereby an intact single-copy transgene can be inserted into a predetermined chromosomal locus with high efficiency (up to 40%), and faithfully transmitted through generations. This system allows high-level global transgene expression or tissue-specific expression depending on the promoter used, or inducible expression such as induced by tetracycline or doxycycline. Using this approach, site-specific transgenic mice can be generated as fast as in 3 months. The technique presented here greatly facilitates murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Fogg PCM, Colloms S, Rosser S, Stark M, Smith MCM (2014) New applications for phage integrases. J Mol Biol 426(15):2703–2716

    Article  CAS  Google Scholar 

  2. Calos MP (2016) Phage integrases for genome editing. Genome Editing Part of the series. In: Advances in experimental medicine and biology. Springer, New York, pp 81–91 Date: 04 March 2016. Series ISSN 0065-2598

    Google Scholar 

  3. Groth AC, Olivares EC, Thyagarajan B, Calos MP (2000) A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci U S A 97:5995–6000

    Article  CAS  Google Scholar 

  4. Keravala A, Groth AC, Jarrahian S, Thyagarajan B, Hoyt JJ, Kirby PJ, Calos MP (2006) A diversity of serine phage integrases mediate site-specific recombination in mammalian cells. Mol Gen Genomics 276:135–146

    Article  CAS  Google Scholar 

  5. Ma QW, Sheng HQ, Yan JB, Cheng S, Huang Y, Chen-Tsai Y, Ren ZR, Huang SZ, Zeng YT (2006) Identification of pseudo attP sites for phage phiC31 integrase in bovine genome. Biochem Biophys Res Commun 345:984–988

    Article  CAS  Google Scholar 

  6. Thorpe HM, Smith MC (1998) In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci U S A 95:5505–5510

    Article  CAS  Google Scholar 

  7. Olivares EC, Hollis RP, Chalberg TW, Meuse L, Kay MA, Calos MP (2002) Site-specific genomic integration produces therapeutic Factor IX levels in mice. Nat Biotechnol 20:1124–1128

    Article  CAS  Google Scholar 

  8. Hollis RP, Stoll SM, Sclimenti CR, Lin J, Chen-Tsai Y, Calos MP (2003) Phage integrases for the construction and manipulation of transgenic mammals. Reprod Biol Endocrinol 1:79

    Article  Google Scholar 

  9. 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. Proc Natl Acad Sci U S A 108:7902–7907

    Article  CAS  Google Scholar 

  10. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70–71

    Article  CAS  Google Scholar 

  11. Hippenmeyer S, Youn YH, Moon HM, Miyamichi K, Zong H, Wynshaw-Boris A, Luo L (2010) Genetic mosaic dissection of Lis1 and Ndel1 in neuronal migration. Neuron 68:695–709

    Article  CAS  Google Scholar 

  12. Fan X, Petitt M, Gamboa M Huang M, Dhal S, Druzin ML, Wu JC, Chen-Tsai Y, Nayak NR (2012) Transient, inducible, placenta-specific gene expression in mice. Endocrinology 153:5637–5644

    Article  CAS  Google Scholar 

  13. Guenther CA, Tasic B, Luo L, Bedell MA (2014) Kingsley DM (2014) A molecular basis for classic blond hair color in Europeans. Nat Genet 46(7):748–752

    Article  CAS  Google Scholar 

  14. Feng D, Dai S, Liu F, Ohtake Y, Zhou Z, Wang H et al (2016) Cre-inducible human CD59 mediates rapid cell ablation after intermedilysin administration. J Clin Invest 126(6):2321–2333

    Article  Google Scholar 

  15. Li K, Wang F, Cao WB, Lv XX, Hua F, Cui B, Zhang XW, Shang S et al (2017) TRIB3 promotes APL progression through stabilization of the oncoprotein PML-RARa ad inhibition of p53-mediated senescence. Cancer Cell 31(5):697–710

    Article  CAS  Google Scholar 

  16. Zhu F, Gamboa M, Farruggio AP, Hippenmeyer S, Tasic B, Shule B, Chen-Tsai Y, Calos MP (2014) Dice, an efficient system for iterative genomic editing in human pluripotent stem cells. Nucleic Acids Res 42(5):e34

    Article  CAS  Google Scholar 

  17. Ruan J, Li H, Xu K, Wu T, Wei J, Zhou R, Liu Z, Mu Y, Yang S-l, Ouyang H, Chen-Tsai RY, Li K (2015) Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs. Sci Rep 5:14253

    Article  Google Scholar 

  18. Booze ML, Hansen JM, Vitiello PF (2016) A novel mouse model for the identification of thioredoxin-1 protein interactions. Free Radic Biol Med 99:533–543

    Article  CAS  Google Scholar 

  19. Sun N, YunJ LJ, Malide D, Liu C, Rovira L et al (2015) Measuring in vivo mitophage. Mol Cell 60(4):685–696

    Article  CAS  Google Scholar 

  20. Devine WP, Wythe JD, George M, Koshiba-Takeuchi K, Bruneau BG (2014) Early patterning and specification of cardiac progenitors in gastrulating mesoderm. elife 3:e03848

    Article  Google Scholar 

  21. Villamizar CA (2014) Characterization of the vascular pathology in the ACTA2 R258C mouse model and cerebrovascular characterization of the ACTA2 null mouse. UT GSBS dissertations and thesis (open acess). Paper 508

    Google Scholar 

  22. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823

    Article  CAS  Google Scholar 

  23. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826

    Article  CAS  Google Scholar 

  24. Wang H, Yang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153(4):910–918

    Article  CAS  Google Scholar 

  25. Quadros RM, Miura H, Harms DW, Akatuka H, Sato T, Aida T, Redder R, Richardson GP et al (2017) Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol 18:92

    Article  Google Scholar 

Download references

Acknowledgment

The author would like to thank all Applied StemCell, Inc. employees who have contributed to improving the TARGATT™ technologies. Thanks also go to our customers who entrusted us on generating TARGATT™ models for their research.

Author information

Authors and Affiliations

  1. Applied StemCell, Inc., Milpitas, CA, USA

    Ruby Yanru Chen-Tsai

Authors
  1. Ruby Yanru Chen-Tsai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ruby Yanru Chen-Tsai .

Editor information

Editors and Affiliations

  1. National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Chengyu Liu

  2. National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Yubin Du

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Chen-Tsai, R.Y. (2019). Using TARGATT™ Technology to Generate Site-Specific Transgenic Mice. In: Liu, C., Du, Y. (eds) Microinjection. Methods in Molecular Biology, vol 1874. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8831-0_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8831-0_4

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8830-3

  • Online ISBN: 978-1-4939-8831-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation