Skip to main content
SpringerLink
Log in
Book cover

Molecular Embryology pp 123–138Cite as

Gene Trapping in Mouse Embryonic Stem Cells

  • Protocol

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 97))

Abstract

Gene trapping in mouse embryonic stem (ES) cells offers a method to create random developmental mutants with a direct route to cloning and defining the expression pattern of the disrupted gene (1). Gene trapping involves the use of reporter gene constructs that are activated following insertion into endogenous transcription units. A number of plasmid- and retroviral-based vectors have been developed, which differ in their requirements for reporter gene activation (reviewed in refs. 2 and 3). “Promoter trap” vectors simply consist of a promoterless reporter gene that is activated following insertions in exons of genes. In contrast, “gene trap” vectors contain a splice acceptor sequence upstream of a reporter and are activated following insertions into introns of genes. Both promoter and gene trap insertions create a fusion transcript from which a portion of the endogenous gene may be readily cloned (4,5). The pattern of reporter gene activity can be monitored in ES-cell derived chimeric embryos (6) or in transgenic embryos following germline transmission (7). With two plasmid-based vectors, reporter gene expression has been shown to reflect accurately that of the endogenous gene (5,8). Ultimately, the function of the trapped gene can be tested following germline transmission. Using this approach, a number of embryonic lethal mutations and visible adult phenotypes have been isolated (4,5,7,9–11).

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   119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

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. Gossler, A., Joyner, A. L., Rossant, J., and Skarnes, W.C. (1989) Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science 244, 463–465.

    Article  PubMed  CAS  Google Scholar 

  2. Karnes, W. C. (1993) The identification of new genes: gene trapping in transgenic mice. Curr. Op. Biotech. 4, 684–689.

    Article  Google Scholar 

  3. Gossler, A. and Zachgo, J. (1993) Gene and enhancer trap screens in ES cell chimaeras, in Gene Targeting: A Practical Approach (Joyner, A., ed.), Oxford University Press, Oxford, pp. 181–227.

    Google Scholar 

  4. von Melchner, H., DeGregori, J. V., Rayburn, H., Reddy, S., Friedel, C., and Ruley, H. E. (1992) Selective disruption of genes expressed in totipotent embryonal stem cells. Genes Dev. 6, 919–927.

    Article  Google Scholar 

  5. Skarnes, W. C., Auerbach, B. A., and Joyner, A. L. (1992) A gene trap approach in mouse embryonic stem cells: the lacZ reporter is activated by splicing, reflects endogenous gene expression, and is mutagenic in mice. Genes Dev. 6, 903–918.

    Article  PubMed  CAS  Google Scholar 

  6. Wurst, W., Rossant, J., Prideaux, V., Kownacka, M., Joyner, A., Hill, D. P., Guillemot, F., Gasca, S., Cado, D., Auerbach, A., and Ang, S.-L. (1995). A large-scale gene-trap screen for insertional mutations in developmentally regulated genes in mice. Genetics 139, 889–899.

    PubMed  CAS  Google Scholar 

  7. Friedrich, G. and Soriano, P. (1991) Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 5, 1513–1523.

    Article  PubMed  CAS  Google Scholar 

  8. Skarnes, W. C., Moss, J. E., Hurtley, S. M., and Beddington, R. S. P. (1995) Capturing genes encoding membrane and secreted proteins important for mouse development. Proc. Natl. Acad. Sci. USA 92, 6592–6596.

    Article  PubMed  CAS  Google Scholar 

  9. DeGregori, J., Russ, A., von Melchner, H., Rayburn, H., Priyaranjan, P., Jenkins, N. A., Copeland, N. G., and Ruley, H. E. (1994) A murine homolog of the yeast RNA1 gene is required for postimplantation development. Genes Dev. 8, 265–276.

    Article  PubMed  CAS  Google Scholar 

  10. Chen, Z., Friedrich, G. A., and Soriano, P. (1994) Transcriptional enhancer factor 1 disruption by a retroviral gene trap leads to heart defects and embryonic lethality in mice. Genes Dev. 8, 2993–2301.

    Google Scholar 

  11. Takeuchi, T, Yamazaki, Y., Katoh-Fuki, Y., Tsuchiya, R., Kondo, S., Motoyama, J., and Higashinakagawa, T. (1995) Gene trap capture of a novel mouse gene jumonji, required for neural tube development. Genes Dev. 9, 1211–1222.

    Article  PubMed  CAS  Google Scholar 

  12. Kerr, W. G., Nolan, G. P., Serafini, A. T., and Herzenberg, L. A. (1989) Transcriptionally defective retroviruses containing lacZ for the in situ detection of endogenous genes and developmentally regulated chromatin. Cold Spring Harbor Symp. Quant. Biol. 54, 767–776.

    Article  PubMed  CAS  Google Scholar 

  13. Mountford, P. S. and Smith, A. G. (1995) Internal ribosome entry sites and dicistronic RNAs in mammalian transgenesis. Trends Genet. 11(5), 179–184.

    Article  PubMed  CAS  Google Scholar 

  14. Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998–9002.

    Article  PubMed  CAS  Google Scholar 

  15. Robertson, E. (ed.) (1987) Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. IRL, Oxford.

    Google Scholar 

  16. Joyner, A. (ed.) (1993) Gene Targeting: A Practical Approach. Oxford University Press, Oxford.

    Google Scholar 

  17. Wassarman, P. M. and DePamphilis, M. L. (eds.) (1993) Methods in Enzymology, vol. 225: Guide to Techniques in Mouse Development. Academic, San Diego, CA.

    Google Scholar 

  18. Hogan, B., Beddington, R., Costanini, F., and Lacy, E. (eds.) (1994) Manipulating the Mouse Embryo. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  19. Nichols, J., Evans, E. P., and Smith, A. G. (1990) Establishment of germline-competent embryonic stem (ES) cells using differentiation inhibiting activity. Development 110, 1341–1348.

    PubMed  CAS  Google Scholar 

  20. Maniatis, T., Fritsch, E. F., and Sambrook, J. (eds.) (1982) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 1.21–1.52.

    Google Scholar 

  21. Smith, A. G. (1991) Culture and differentiation of embryonic stem cells. J. Tissue Culture Meth. 13, 89–94.

    Article  Google Scholar 

  22. Ure, J. M., Fiering, S., and Smith, A. G. (1992) A rapid and efficient method for freezing and recovering clones of embryonic stem cells. Trends Genetics 8(1), 6.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. BBSRC Centre for Genome Research, University of Edinburgh, Scotland, UK

    Jane Brennan & William C. Skarnes

Authors
  1. Jane Brennan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. William C. Skarnes
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Dental and Medical Schools of Guy’s, King’s, and St. Thomas’s Hospitals, King’s College, London, UK

    Paul T. Sharpe  & Ivor Mason  & 

Rights and permissions

Reprints and permissions

Copyright information

© 1999 Humana Press Inc.

About this protocol

Cite this protocol

Brennan, J., Skarnes, W.C. (1999). Gene Trapping in Mouse Embryonic Stem Cells. In: Sharpe, P.T., Mason, I. (eds) Molecular Embryology. Methods in Molecular Biology™, vol 97. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-270-8:123

Download citation

  • DOI: https://doi.org/10.1385/1-59259-270-8:123

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-0-89603-387-0

  • Online ISBN: 978-1-59259-270-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation