Ends-out, or replacement, gene targeting in <em>Drosophila</em>

Wei Gong

Kent Golic

Stowers Institute for Medical Research, Kansas City, MO 64110

^{Present address: Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112.}

^{To whom correspondence should be addressed. E-mail:}ude.hatu.ygoloib@cilog.

Edited by Michael S. Levine, University of California, Berkeley, CA, and approved January 8, 2003

Received 2002 Aug 14

Abstract

Ends-in and ends-out refer to the two arrangements of donor DNA that can be used for gene targeting. Both have been used for targeted mutagenesis, but require donors of differing design. Ends-out targeting is more frequently used in mice and yeast because it gives a straightforward route to replace or delete a target locus. Although ends-in targeting has been successful in Drosophila, an attempt at ends-out targeting failed. To test whether ends-out targeting could be used in Drosophila, we applied two strategies for ends-out gene replacement at the endogenous yellow (y) locus in Drosophila. First, a mutant allele was rescued by replacement with an 8-kb y⁺ DNA fragment at a rate of ≈1/800 gametes. Second, a wild-type gene was disrupted by the insertion of a marker gene in exon 1 at a rate of ≈1/380 gametes. The I-SceI endonuclease component alone is not sufficient for targeting: the FLP recombinase is also needed to generate the extrachromosomal donor. When both components are used we find that ends-out targeting can be approximately as efficient as ends-in targeting, and is likely to be generally useful for Drosophila gene targeting.

Abstract

Gene targeting is the modification of an endogenous gene sequence by recombination between an introduced DNA fragment and the homologous target gene. Over the past 25 years, gene targeting has been widely used in model eukaryotes, first in yeast and then in mice (1, 2), but the difficulty of introducing a linear DNA molecule into germ-line cells hindered its development for Drosophila. Recently, a method to generate such a linear fragment in vivo was reported, accompanied by a demonstration of ends-in or insertional gene targeting (3). This occurs when a DNA double-strand break (DSB) is made in a donor DNA fragment within a stretch of DNA that is homologous to the target locus, and results in the insertion of the donor to generate a duplication of the targeted region. An alternative arrangement, where DSBs are provided at each end of a homologous segment, is termed ends-out targeting, and causes a segment of chromosome to be replaced with an introduced segment (Fig. (Fig.1).1). In mouse and in yeast some studies show that ends-out targeting is less efficient than ends-in (4–6), whereas others indicate that the two types can be equally efficient (7, 8). Doubts about the efficacy of ends-out targeting in Drosophila have been raised because of a previous failure (9). We undertook this work to determine whether ends-out targeting could be usefully implemented in Drosophila.

An external file that holds a picture, illustration, etc.
Object name is pq0535280001.jpg

Figure 1

Two general forms of gene targeting. Donor DNA molecules are diagramed above their targets, along with the expected products of recombination. The light gray region is the target-homologous DNA; the black region is the positive marker gene.

n, number of vials scored for targeting; T, number of vials with targeting events, which is taken to be the minimum estimate of independent targeting events; NT, number of vials with nontargeted events.

Acknowledgments

We thank Yikang Rong, Simon Titen, Mary Golic, and Keith Maggert for help and advice during this work. This work was supported by National Institutes of Health Grants GM60700 and GM65604 and by the Stowers Institute for Medical Research.

Acknowledgments

Abbreviations

DSB	double-strand break
FRT	FLP recombination target

Abbreviations

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

Footnotes

References

1. Rothstein R. Methods Enzymol. 1991;194:281–301.[PubMed]
2. Muller U. Mech Dev. 1999;82:3–21.[PubMed]
3. Rong Y S, Golic K G. Science. 2000;288:2013–2018.[PubMed]
4. Hasty P, Rivera-Pérez J, Chang C, Bradley A. Mol Cell Biol. 1991;11:4509–4517.
5. Hastings P J, McGill C, Shafer B, Strathern J N. Genetics. 1993;135:973–980.
6. Leung W-Y, Malkova A, Haber J E. Genetics. 1997;94:6851–6856.
7. Thomas K R, Capecchi M R. Cell. 1987;51:503–512.[PubMed]
8. Deng C, Capecchi M R. Mol Cell Biol. 1992;12:3365–3371.
9. Bellaiche Y, Mogila V, Perrimon N. Genetics. 1999;152:1037–1044.
10. Klemenz R, Weber U, Gehring W J. Nucleic Acids Res. 1987;15:3947–3959.
11. Geyer P K, Corces V G. Genes Dev. 1987;1:996–1004.[PubMed]
12. Rubin G M, Spradling A C. Nucleic Acids Res. 1983;11:6341–6351.
13. Golic K G, Lindquist S. Cell. 1989;59:499–509.[PubMed]
14. Rubin G M, Spradling A C. Science. 1982;218:348–353.[PubMed]
15. Orr-Weaver T, Szostak J W, Rothstein R J. Proc Natl Acad Sci USA. 1981;78:6354–6358.
16. Rong Y S, Titen S W, Xie H B, Golic M M, Bastiani M, Bandyopahdyay P, Olivera B M, Brodsky M, Rubin G M, Golic K G. Genes Dev. 2002;16:1568–1581.
17. Scwartzberg P L, Robertson E, Goff S P. Proc Natl Acad Sci USA. 1990;87:3210–3214.
18. Thomas K R, Deng C, Capecchi M R. Mol Cell Biol. 1992;12:2919–2923.
19. Rong Y S, Golic K G. Genetics. 2001;157:1307–1312.
20. Seum C, Pauli D, Delattre M, Jaquet Y, Spierer A, Spierer P. Genetics. 2002;161:1125–1136.
21. Michiels F, Gasch A, Kaltschmidt B, Renkawitz-Pohl R. EMBO J. 1989;8:1559–1565.
22. Bonner J J, Parks C, Parker-Thornburg J, Mortin M A, Pelham H R B. Cell. 1984;37:979–991.[PubMed]
23. Golic M M, Golic K G. Genetics. 1996;143:385–400.
24. Golic M M, Rong Y S, Petersen R B, Lindquist S L, Golic K G. Nucleic Acids Res. 1997;25:3665–3671.
25. Dale E C, Ow D W. Proc Natl Acad Sci USA. 1991;88:10558–10562.
26. Gu H, Marth J D, Orban P C, Mossman H, Rajewsky K. Science. 1994;265:103–106.[PubMed]
27. Meyers E N, Lewandowski M, Martin G R. Nat Genet. 1998;18:136–141.[PubMed]
28. Bunting M, Bernstein K E, Greer J M, Capecchi M R, Thomas K R. Genes Dev. 1999;13:1524–1528.
29. Siegal M L, Hartl D L. Genetics. 1996;144:715–726.
30. Surosky R, Tye B-K. Proc Natl Acad Sci USA. 1985;82:2106–2110.
31. Matzuk M M, Finegold M J, Su J G, Hsueh A J, Bradley A. Nature. 1992;360:313–319.[PubMed]
32. Mansour S L, Thomas K R, Capecchi M R. Nature. 1988;336:348–352.[PubMed]