Abstract
DNA double-strand breaks provide a powerful means to modify the genome. This chapter describes how to generate and use these breaks to target specific sequences, or other modifications to the Drosophila genome. Both P element dependent gene conversion, in which the chromosomal DNA is broken, and the Rong and Golic gene-targeting technique, in which the targeting vector contains the DNA break are explained. The strengths and limitations of both methods are presented so that the user can choose the appropriate method for their particular situation. The efficiency of both methods depends upon the genomic location being modified, although few, if any, genomic locations are refractory to either method. It cannot be emphasized strongly enough that the investigator should be prepared to invest sufficient time into setting up and running these experiments properly.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., Gocayne, J. D., Amanatides, P. G., et al. (2000) The genome sequence of Drosophila melanogaster. Science 287, 2185–2195.
Rubin, G. M. and Lewis, E. B. (2000) A brief history of Drosophila’s contributions to genome research. Science 287, 2216–2218.
Kornberg, T. B. and Krasnow, M. A. (2000) The Drosophila genome sequence: implications for biology and medicine. Science 287, 2218–2220.
Gloor, G. B., Nassif, N. A., Johnson-Schlitz, D. M., Preston, C. R., and Engels, W. R. (1991) Targeted gene replacement in Drosophila via P element-induced gap repair. Science 253, 1110–1117.
Nassif, N., Penney, J., Pal, S., Engels, W. R., and Gloor, G. B. (1994) Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair. Trends Biochem. Sci. 14, 1613–1625.
Paques, F. and Haber, J. E. (1999) Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349–404.
Nassif, N. and Engels, W. (1993) DNA homology requirements for mitotic gap repair in Drosophila. Proc. Natl. Acad. Sci. USA 90, 1262–1266.
Johnson-Schlitz, D. M. and Engels, W. R. (1993) P-element-induced interallelic gene conversion of insertions and deletions in Drosophila melanogaster. Mol. Cell. Biol. 13, 7006–7018.
Engels, W. R., Johnson-Schlitz, D. M., Eggleston, W. B., and Sved, J. (1990) High-frequency P element loss in Drosophila is homolog dependent. Cell 62, 515–525.
Keeler, K. J., Dray, T., Penney, J. E., and Gloor, G. B. (1996) Gene targeting of a plasmid-borne sequence to a double-strand DNA break in Drosophila melanogaster. Mol. Cell. Biol. 16, 522–528.
Engels, W. R., Preston, C. R., and Johnson-Schlitz, D. M. (1994) Long-range cis preference in DNA homology search over the length of a Drosophila chromosome. Science 263, 1623–1625.
Keeler, K. J. and Gloor, G. B. (1997) Efficient gap repair in Drosophila melanogaster requires a maximum of 31 nucleotides of homologous sequence at the searching ends. Mol. Cell. Biol. 17, 627–634.
Morris, J. R., Geyer, P. K., and Wu, C. T. (1999) Core promoter elements can regulate transcription on a separate chromosome in trans. Genes Dev. 13, 253–258.
Merli, C., Bergstrom, D. E., Cygan, J. A., and Blackman, R. K. (1996) Promoter specificity mediates the independent regulation of neighboring genes. Genes Dev. 10, 1260–1270.
McCall, K. and Bender, W. (1996) Probes of chromatin accessibility in the Drosophila bithorax complex respond differently to Polycomb-mediated repression. EMBO J. 15, 569–580.
Williams, C. J. and O’Hare, K. (1996) Elimination of introns at the Drosophila suppressor-of-forked locus by P-element-mediated gene conversion shows that an RNA lacking a stop codon is dispensable. Genetics 143, 345–351.
Lankenau, D. H., Corces, V. G., and Engels, W. R. (1996) Comparison of targeted-gene replacement frequencies in Drosophila melanogaster at the forked and white loci. Mol. Cell. Biol. 16, 3535–3544.
Rong, Y. S. and Golic, K. G. (2000) Gene targeting by homologous recombination in Drosophila [see comments]. Science 288, 2013–2018.
Spradling, A. C. and Rubin, G. M. (1982) Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218, 341–347.
Hastings, P. J., McGill, C., Shafer, B., and Strathern, J. N. (1993) Ends-in vs. ends-out recombination in yeast. Genetics 135, 973–980.
Rong, Y. and Golic, K. (2001) A targeted gene knockout in Drosophila. Genetics 157, 1307–1312.
Dray, T. and Gloor, G. B. (1997) Homology requirements for targeting heterologous sequences during P-induced gap repair in Drosophila melanogaster. Genetics 147, 689–699.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Gloor, G.B. (2004). Gene Targeting in Drosophila . In: Miller, W.J., Capy, P. (eds) Mobile Genetic Elements. Methods in Molecular Biology, vol 260. Humana Press. https://doi.org/10.1385/1-59259-755-6:097
Download citation
DOI: https://doi.org/10.1385/1-59259-755-6:097
Publisher Name: Humana Press
Print ISBN: 978-1-58829-007-6
Online ISBN: 978-1-59259-755-0
eBook Packages: Springer Protocols