Skip to main content
SpringerLink
Log in
Book cover

Xenopus Protocols pp 129–141Cite as

Targeted Gene Disruption with Engineered Zinc-Finger Nucleases (ZFNs)

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 917))

Abstract

Reverse genetics in Xenopus has been limited to knockdown strategies using antisense morpholino oligonucleotides (MOs). Recently, engineered zinc-finger nucleases have been used to induce targeted mutations resulting in null alleles. Zinc-finger nuclease (ZFN) technology has been adapted to induce null mutations in many systems previously refractory to targeted gene inactivation. Here we provide a general protocol for inducing targeted mutations in Xenopus tropicalis using ZFNs, a method to detect resulting mutations, and the steps to generate homozygous mutant embryos.

This is a preview of subscription content, 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   99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   199.99
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Bibikova M, Golic M, Golic KG, Carroll D (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161(3):1169–1175

    PubMed  CAS  Google Scholar 

  2. Beumer K, Bhattacharyya G, Bibikova M, Trautman JK, Carroll D (2006) Efficient gene targeting in Drosophila with zinc-finger nucleases. Genetics 172(4):2391–2403

    Article  PubMed  CAS  Google Scholar 

  3. Doyon Y, McCammon JM, Miller JC, Faraji F, Ngo C, Katibah GE, Amora R, Hocking TD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Amacher SL (2008) Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol 26(6):702–708

    Article  PubMed  CAS  Google Scholar 

  4. Meng X, Noyes MB, Zhu LJ, Lawson ND, Wolfe SA (2008) Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat Biotechnol 26(6):695–701

    Article  PubMed  CAS  Google Scholar 

  5. Foley JE, Yeh JR, Maeder ML, Reyon D, Sander JD, Peterson RT, Joung JK (2009) Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by oligomerized pool engineering (OPEN). PLoS One 4(2):e4348

    Article  PubMed  Google Scholar 

  6. Siekmann AF, Standley C, Fogarty KE, Wolfe SA, Lawson ND (2009) Chemokine signaling guides regional patterning of the first embryonic artery. Genes Dev 23(19):2272–2277

    Article  PubMed  CAS  Google Scholar 

  7. Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O, Wang N, Lee G, Bartsevich VV, Lee YL, Guschin DY, Rupniewski I, Waite AJ, Carpenito C, Carroll RG, Orange JS, Urnov FD, Rebar EJ, Ando D, Gregory PD, Riley JL, Holmes MC, June CH (2008) Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol 26(7):808–816

    Article  PubMed  CAS  Google Scholar 

  8. Santiago Y, Chan E, Liu PQ, Orlando S, Zhang L, Urnov FD, Holmes MC, Guschin D, Waite A, Miller JC, Rebar EJ, Gregory PD, Klug A, Collingwood TN (2008) Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci U S A 105(15):5809–5814

    Article  PubMed  CAS  Google Scholar 

  9. Cost GJ, Freyvert Y, Vafiadis A, Santiago Y, Miller JC, Rebar E, Collingwood TN, Snowden A, Gregory PD (2009) BAK and BAX deletion using zinc-finger nucleases yields apoptosis-resistant CHO cells. Biotechnol Bioeng 105(2):330–340

    Article  Google Scholar 

  10. Liu PQ, Chan EM, Cost GJ, Zhang L, Wang J, Miller JC, Guschin DY, Reik A, Holmes MC, Mott JE, Collingwood TN, Gregory PD (2010) Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases. Biotechnol Bioeng 106(1):97–105

    PubMed  CAS  Google Scholar 

  11. Malphettes L, Freyvert Y, Chang J, Liu PQ, Chan E, Miller JC, Zhou Z, Nguyen T, Tsai C, Snowden AW, Collingwood TN, Gregory PD, Cost GJ (2010) Highly efficient deletion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies. Biotechnol Bioeng 106(5):774–783

    Article  PubMed  CAS  Google Scholar 

  12. Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui X, Meng X, Vincent A, Lam S, Michalkiewicz M, Schilling R, Foeckler J, Kalloway S, Weiler H, Menoret S, Anegon I, Davis GD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jacob HJ, Buelow R (2009) Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325(5939):433

    Article  PubMed  CAS  Google Scholar 

  13. Mashimo T, Takizawa A, Voigt B, Yoshimi K, Hiai H, Kuramoto T, Serikawa T (2010) Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID) using zinc-finger nucleases. PLoS One 5(1):e8870

    Article  PubMed  Google Scholar 

  14. Young JJ, Cherone JM, Doyon Y, Ankoudinova I, Faraji FM, Lee AH, Ngo C, Guschin DY, Paschon DE, Miller JC, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Harland RM, Zeitler B (2011) Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases. Proc Natl Acad Sci U S A 108(17):7052–7057

    Article  PubMed  CAS  Google Scholar 

  15. Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA, Gregory PD, Pabo CO, Rebar EJ (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25(7):778–785

    Article  PubMed  CAS  Google Scholar 

  16. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11(9):636–646

    Article  PubMed  CAS  Google Scholar 

  17. Maeder ML, Thibodeau-Beganny S, Sander JD, Voytas DF, Joung JK (2009) Oligomerized pool engineering (OPEN): an ‘open-source’ protocol for making customized zinc-finger arrays. Nat Protoc 4(10):1471–1501

    Article  PubMed  CAS  Google Scholar 

  18. Kim HJ, Lee HJ, Kim H, Cho SW, Kim JS (2009) Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome Res 19(7):1279–1288

    Article  PubMed  CAS  Google Scholar 

  19. McCammon JM, Doyon Y, Amacher SL (2011) Inducing high rates of targeted mutagenesis in zebrafish using zinc finger nucleases (ZFNs). Methods Mol Biol 770:505–527

    Article  PubMed  CAS  Google Scholar 

  20. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326(5959):1509–1512

    Article  PubMed  CAS  Google Scholar 

  21. Moscou MJ, Bogdanove AJ (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326(5959):1501

    Article  PubMed  CAS  Google Scholar 

  22. Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, Cost GJ, Zhang L, Santiago Y, Miller JC, Zeitler B, Cherone JM, Meng X, Hinkley SJ, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R (2011) Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 29(8):731–734

    Article  PubMed  CAS  Google Scholar 

  23. Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186(2):757–761

    Article  PubMed  CAS  Google Scholar 

  24. Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29(2):143–148

    Article  PubMed  CAS  Google Scholar 

  25. Turner DL, Weintraub H (1994) Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev 8(12):1434–1447

    Article  PubMed  CAS  Google Scholar 

  26. Grammer TC, Khokha MK, Lane MA, Lam K, Harland RM (2005) Identification of mutants in inbred Xenopus tropicalis. Mech Dev 122(3):263–272

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We wish to thank the Conlon and Moens labs for the celery extract preparation protocols.

Author information

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA

    John J. Young & Richard M. Harland

Authors
  1. John J. Young
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Richard M. Harland
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John J. Young .

Editor information

Editors and Affiliations

  1. , Institute of Medical Sciences, University of Aberdeen, FORESTERHILL, ABERDEEN, AB25 2ZD, United Kingdom

    STEFAN HOPPLER

  2. , Department of Biological Science, University of Calgary, University Drive NW 2500, Calgary, T2N 1N4, Canada

    Peter D Vize

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Young, J.J., Harland, R.M. (2012). Targeted Gene Disruption with Engineered Zinc-Finger Nucleases (ZFNs). In: HOPPLER, S., Vize, P. (eds) Xenopus Protocols. Methods in Molecular Biology, vol 917. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-992-1_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-992-1_7

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-991-4

  • Online ISBN: 978-1-61779-992-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation