Skip to main content
SpringerLink
Log in

Chimeric Mouse Generation by ES Cell Blastocyst Microinjection and Uterine Transfer

  • Protocol
  • First Online:
Book cover Microinjection

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

Abstract

The ability to generate chimeric mice through microinjecting embryonic stem (ES) cells into blastocysts is a critical step for the conventional ES cell-mediated knockout technology. In recent years, designer nuclease-based methods, especially the CRISPR technology, have substantially decreased the needs for blastocyst microinjection. However, this method has still remained as a valuable technique for generating sophisticated genetic models as well as for stem cell research. In this chapter, we describe the detailed procedures used in our laboratory on how to use ES cells to produce chimeric mice, including derivation and inactivation of MEF feeder cells, culturing and handling of mouse ES cells, collection and microinjection of blastocysts, and finally implantation of injected blastocysts into the uteri of pseudopregnant surrogate mothers.

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. Smithies O, Connell GE, Dixon GH (1962) Chromosomal rearrangements and the evolution of haptoglobin genes. Nature 196:232–236

    Article  CAS  Google Scholar 

  2. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156

    Article  CAS  Google Scholar 

  3. Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlapati RS (1985) Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature 317:230–234

    Article  CAS  Google Scholar 

  4. Thomas KR, Capecchi MR (1986) Introduction of homologous DNA sequences into mammalian cells induces mutations in the cognate gene. Nature 324:34–38. https://doi.org/10.1038/324034a0

    Article  CAS  PubMed  Google Scholar 

  5. Papaioannou VE, McBurney MW, Gardner RL, Evans MJ (1975) Fate of teratocarcinoma cells injected into early mouse embryos. Nature 258:70–73

    Article  CAS  Google Scholar 

  6. Mansour SL, Thomas KR, Capecchi MR (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336:348–352. https://doi.org/10.1038/336348a0

    Article  CAS  PubMed  Google Scholar 

  7. Orban PC, Chui D, Marth JD (1992) Tissue- and site-specific DNA recombination in transgenic mice. Proc Natl Acad Sci U S A 89:6861–6865

    Article  CAS  Google Scholar 

  8. Gu H, Zou YR, Rajewsky K (1993) Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 73:1155–1164

    Article  CAS  Google Scholar 

  9. Austin CP, Battey JF, Bradley A, Bucan M, Capecchi M, Collins FS, Dove WF, Duyk G, Dymecki S, Eppig JT, Grieder FB, Heintz N, Hicks G, Insel TR, Joyner A, Koller BH, Lloyd KC, Magnuson T, Moore MW, Nagy A, Pollock JD, Roses AD, Sands AT, Seed B, Skarnes WC, Snoddy J, Soriano P, Stewart DJ, Stewart F, Stillman B, Varmus H, Varticovski L, Verma IM, Vogt TF, von Melchner H, Witkowski J, Woychik RP, Wurst W, Yancopoulos GD, Young SG, Zambrowicz B (2004) The knockout mouse project. Nat Genet 36:921–924. https://doi.org/10.1038/ng0904-921

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wood SA, Allen ND, Rossant J, Auerbach A, Nagy A (1993) Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 365:87–89. https://doi.org/10.1038/365087a0

    Article  CAS  PubMed  Google Scholar 

  11. Valenzuela DM, Murphy AJ, Frendewey D, Gale NW, Economides AN, Auerbach W, Poueymirou WT, Adams NC, Rojas J, Yasenchak J, Chernomorsky R, Boucher M, Elsasser AL, Esau L, Zheng J, Griffiths JA, Wang X, Su H, Xue Y, Dominguez MG, Noguera I, Torres R, Macdonald LE, Stewart AF, DeChiara TM, Yancopoulos GD (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat Biotechnol 21:652–659. https://doi.org/10.1038/nbt822

    Article  CAS  PubMed  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 325:433. https://doi.org/10.1126/science.1172447

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sung YH, Baek IJ, Kim DH, Jeon J, Lee J, Lee K, Jeong D, Kim JS, Lee HW (2013) Knockout mice created by TALEN-mediated gene targeting. Nat Biotechnol 31:23–24. https://doi.org/10.1038/nbt.2477

    Article  CAS  PubMed  Google Scholar 

  14. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918. https://doi.org/10.1016/j.cell.2013.04.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Shen B, Zhang J, Wu H, Wang J, Ma K, Li Z, Zhang X, Zhang P, Huang X (2013) Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res 23:720–723. https://doi.org/10.1038/cr.2013.46

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Eggan K, Akutsu H, Loring J, Jackson-Grusby L, Klemm M, Rideout WM 3rd, Yanagimachi R, Jaenisch R (2001) Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc Natl Acad Sci U S A 98:6209–6214. https://doi.org/10.1073/pnas.101118898

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kobayashi T, Yamaguchi T, Hamanaka S, Kato-Itoh M, Yamazaki Y, Ibata M, Sato H, Lee YS, Usui J, Knisely AS, Hirabayashi M, Nakauchi H (2010) Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell 142:787–799. https://doi.org/10.1016/j.cell.2010.07.039

    Article  CAS  Google Scholar 

  18. Stewart CL (1993) Production of chimeras between embryonic stem cells and embryos. Methods Enzymol 225:823–855

    Article  CAS  Google Scholar 

  19. Schuster-Gossler K, Lee AW, Lerner CP, Parker HJ, Dyer VW, Scott VE, Gossler A, Conover JC (2001) Use of coisogenic host blastocysts for efficient establishment of germline chimeras with C57BL/6J ES cell lines. BioTechniques 31:1022–1024 1026

    Article  CAS  Google Scholar 

  20. Pettitt SJ, Liang Q, Rairdan XY, Moran JL, Prosser HM, Beier DR, Lloyd KC, Bradley A, Skarnes WC (2009) Agouti C57BL/6N embryonic stem cells for mouse genetic resources. Nat Methods 6:493–495. https://doi.org/10.1038/nmeth.1342

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Cui L, Zhang Z, Sun F, Duan X, Wang M, Di K, Li X (2014) Transcervical embryo transfer in mice. J Am Assoc Lab Anim Sci 53:228–231

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute at the US National Institutes of Health.

Author information

Authors and Affiliations

  1. Transgenic Core Facility, Division of Intramural Research, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Yubin Du, Wen Xie, Fan Zhang & Chengyu Liu

Authors
  1. Yubin Du
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wen Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chengyu Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chengyu Liu .

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

Du, Y., Xie, W., Zhang, F., Liu, C. (2019). Chimeric Mouse Generation by ES Cell Blastocyst Microinjection and Uterine Transfer. 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_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8831-0_6

  • 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