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.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
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.
Karnes, W. C. (1993) The identification of new genes: gene trapping in transgenic mice. Curr. Op. Biotech. 4, 684–689.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Mountford, P. S. and Smith, A. G. (1995) Internal ribosome entry sites and dicistronic RNAs in mammalian transgenesis. Trends Genet. 11(5), 179–184.
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.
Robertson, E. (ed.) (1987) Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. IRL, Oxford.
Joyner, A. (ed.) (1993) Gene Targeting: A Practical Approach. Oxford University Press, Oxford.
Wassarman, P. M. and DePamphilis, M. L. (eds.) (1993) Methods in Enzymology, vol. 225: Guide to Techniques in Mouse Development. Academic, San Diego, CA.
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.
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.
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.
Smith, A. G. (1991) Culture and differentiation of embryonic stem cells. J. Tissue Culture Meth. 13, 89–94.
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights 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