Target-Selected Inactivation of the Zebrafish rag1 Gene

American Association for the Advancement of Science (AAAS) - Tập 297 Số 5578 - Trang 99-102 - 2002
Erno Wienholds1, Stefan Schulte‐Merker2, Brigitte Walderich2, Ronald H.A. Plasterk1
1Hubrecht Laboratory, Center for Biomedical Genetics, Uppsalalaan 8, 3584 CT, Utrecht, Netherlands.
2ARTEMIS Pharmaceuticals GmbH/ Exelixis Deutschland, Spemannstrasse 35, 72076 Tübingen, Germany.

Tóm tắt

The zebrafish has become a favorite organism for genetic analysis of vertebrate development, but methods for generating mutants by reverse genetic approaches have been lacking. We report a method to obtain stable mutants of a gene based on knowledge of the gene sequence only. Parental fish were mutagenized with N -ethyl- N -nitrosourea; in 2679 F 1 fish, the rag1 gene was analyzed for heterozygous mutations by resequencing. In total, we found 15 mutations: 9 resulted in amino acid substitutions and 1 resulted in a premature stop codon. This truncation mutant was found to be homozygous viable and defective in V(D)J joining. Although presumably immune deficient, these homozygous rag1 mutant fish are able to reach adulthood and are fertile. As sperm samples from all 2679 F 1 fish were collected and cryopreserved, we have in principle generated a mutant library from which mutants of most zebrafish genes can be isolated.

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Tài liệu tham khảo

Haffter P., et al., Development 123, 1 (1996).

Driever W., et al., Development 123, 37 (1996).

Hukriede N., et al., Genome Res. 11, 2127 (2001).

Geisler R., et al., Nature Genet. 23, 86 (1999).

Woods I. G., et al., Genome Res. 10, 1903 (2000).

Nasevicius A., Ekker S. C., Nature Genet. 26, 216 (2000).

10.1126/science.2660260

Thompson S., Clarke A. R., Pow A. M., Hooper M. L., Melton D. W., Cell 56, 313 (1989).

Ma C., Fan L., Ganassin R., Bols N., Collodi P., Proc. Natl. Acad. Sci. U.S.A 98, 2461 (2001).

Jansen G., et al., Nature Genet. 21, 414 (1999).

Liu L. X., et al., Genome Res. 9, 859 (1999).

Materials and methods are available as supporting material on Science Online.

Willett C. E., Cherry J. J., Steiner L. A., Immunogenetics 45, 394 (1997).

10.1101/gr.8.3.186

Gordon D., Desmarais C., Green P., Genome Res. 11, 614 (2001).

10.1093/nar/25.14.2745

10.1101/gr.8.3.195

E. Wienholds S. Schulte-Merker B Walderich R. H. A. Plasterk data not shown.

Willett C. E., Zapata A. G., Hopkins N., Steiner L. A., Dev. Biol. 182, 331 (1997).

10.1016/0092-8674(89)90760-5

N. Danilova and L. Steiner personal communication.

Danilova N., Hohman V. S., Kim E. H., Steiner L. A., Immunogenetics 52, 81 (2000).

Ballinger D. G., Benzer S., Proc. Natl. Acad. Sci. U.S.A. 86, 9402 (1989).

Kaiser K., Goodwin S. F., Proc. Natl. Acad. Sci. U.S.A. 87, 1686 (1990).

Bentley A., MacLennan B., Calvo J., Dearolf C. R., Genetics 156, 1169 (2000).

Zwaal R. R., Broeks A., van Meurs J., Groenen J. T., Plasterk R. H., Proc. Natl. Acad. Sci. U.S.A. 90, 7431 (1993).

10.1038/ng0997-119

Parinov S., Sundaresan V., Curr. Opin. Biotechnol. 11, 157 (2000).

10.1038/74542

Beier D. R., Mamm. Genome 11, 294 (2000).

Coghill E. L., et al., Nature Genet. 19, 19 (2002).

van Eeden F. J., Granato M., Odenthal J., Haffter P., Methods Cell Biol. 60, 21 (1999).

Notarangelo L. D., Villa A., Schwarz K., Curr. Opin. Immunol. 11, 435 (1999).

Kulkosky J., Jones K. S., Katz R. A., Mack J. P., Skalka A. M., Mol. Cell Biol. 12, 2331 (1992).

We thank R. Nordin I. Santana-Stamm H. Geiger D. Labusch C. Weiler and C. Weiler for help with testis freezing in vitro fertilization and fin clipping and the Tubingen 2000 screen consortium; S. Wicks F. Verbeek and E. Berezikov for help with bioinformatics; D. Jongejan-Zivkovic F. van Eeden E. Cuppen and G. Stott for stimulating discussions and help with preparation of the manuscript; and L. Steiner for sharing unpublished results and providing primer sequences. R.H.A.P. is supported by a SPINOZA grant from the Netherlands Organization for Scientific Research. Supporting Online Material www.sciencemag.org/cgi/content/full/297/5578/99/DC1 Materials and Methods