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. 1996 Oct;144(2):715–726. doi: 10.1093/genetics/144.2.715

Transgene Coplacement and High Efficiency Site-Specific Recombination with the Cre/Loxp System in Drosophila

M L Siegal 1, D L Hartl 1
PMCID: PMC1207562  PMID: 8889532

Abstract

Studies of gene function and regulation in transgenic Drosophila are often compromised by the possibility of genomic position effects on gene expression. We have developed a method, called transgene coplacement, in which any two sequences can be positioned at exactly the same site and orientation in the genome. Transgene coplacement makes use of the bacteriophage P1 system of Cre/loxP site-specific recombination, which we have introduced into Drosophila. In the presence of a cre transgene driven by a dual hsp70-Mos1 promoter, a white reporter gene flanked by loxP sites is excised with virtually 100% efficiency both in somatic cells and in germ cells. A strong maternal effect, resulting from Cre recombinase present in the oocyte, is observed as white or mosaic eye color in F(1) progeny. Excision in germ cells of the F(1) yields a strong grand-maternal effect, observed as a highly skewed ratio of eye-color phenotypes in the F(2) generation. The excision reactions of Cre/loxP and the related FLP/FRT system are used to create Drosophila lines in which transgenes are at exactly allelic sites in homologous chromosomes.

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Selected References

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  1. Albert H., Dale E. C., Lee E., Ow D. W. Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J. 1995 Apr;7(4):649–659. doi: 10.1046/j.1365-313x.1995.7040649.x. [DOI] [PubMed] [Google Scholar]
  2. Austin S., Ziese M., Sternberg N. A novel role for site-specific recombination in maintenance of bacterial replicons. Cell. 1981 Sep;25(3):729–736. doi: 10.1016/0092-8674(81)90180-x. [DOI] [PubMed] [Google Scholar]
  3. Baubonis W., Sauer B. Genomic targeting with purified Cre recombinase. Nucleic Acids Res. 1993 May 11;21(9):2025–2029. doi: 10.1093/nar/21.9.2025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brennan M. D., Dickinson W. J. Complex developmental regulation of the Drosophila affinidisjuncta alcohol dehydrogenase gene in Drosophila melanogaster. Dev Biol. 1988 Jan;125(1):64–74. doi: 10.1016/0012-1606(88)90059-0. [DOI] [PubMed] [Google Scholar]
  5. Chou T. B., Perrimon N. Use of a yeast site-specific recombinase to produce female germline chimeras in Drosophila. Genetics. 1992 Jul;131(3):643–653. doi: 10.1093/genetics/131.3.643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fischer J. A., Giniger E., Maniatis T., Ptashne M. GAL4 activates transcription in Drosophila. Nature. 1988 Apr 28;332(6167):853–856. doi: 10.1038/332853a0. [DOI] [PubMed] [Google Scholar]
  7. Fukushige S., Sauer B. Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7905–7909. doi: 10.1073/pnas.89.17.7905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gloor G. B., Nassif N. A., Johnson-Schlitz D. M., Preston C. R., Engels W. R. Targeted gene replacement in Drosophila via P element-induced gap repair. Science. 1991 Sep 6;253(5024):1110–1117. doi: 10.1126/science.1653452. [DOI] [PubMed] [Google Scholar]
  9. Hartl D. L., Nurminsky D. I., Jones R. W., Lozovskaya E. R. Genome structure and evolution in Drosophila: applications of the framework P1 map. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6824–6829. doi: 10.1073/pnas.91.15.6824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hutter C. M., Rand D. M. Competition between mitochondrial haplotypes in distinct nuclear genetic environments: Drosophila pseudoobscura vs. D. persimilis. Genetics. 1995 Jun;140(2):537–548. doi: 10.1093/genetics/140.2.537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Karess R. E., Rubin G. M. Analysis of P transposable element functions in Drosophila. Cell. 1984 Aug;38(1):135–146. doi: 10.1016/0092-8674(84)90534-8. [DOI] [PubMed] [Google Scholar]
  12. Kilby N. J., Snaith M. R., Murray J. A. Site-specific recombinases: tools for genome engineering. Trends Genet. 1993 Dec;9(12):413–421. doi: 10.1016/0168-9525(93)90104-p. [DOI] [PubMed] [Google Scholar]
  13. Konsolaki M., Sanicola M., Kozlova T., Liu V., Arcà B., Savakis C., Gelbart W. M., Kafatos F. C. FLP-mediated intermolecular recombination in the cytoplasm of Drosophila embryos. New Biol. 1992 May;4(5):551–557. [PubMed] [Google Scholar]
  14. Kühn R., Schwenk F., Aguet M., Rajewsky K. Inducible gene targeting in mice. Science. 1995 Sep 8;269(5229):1427–1429. doi: 10.1126/science.7660125. [DOI] [PubMed] [Google Scholar]
  15. Laurie-Ahlberg C. C., Stam L. F. Use of P-element-mediated transformation to identify the molecular basis of naturally occurring variants affecting Adh expression in Drosophila melanogaster. Genetics. 1987 Jan;115(1):129–140. doi: 10.1093/genetics/115.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lohe A. R., Lidholm D. A., Hartl D. L. Genotypic effects, maternal effects and grand-maternal effects of immobilized derivatives of the transposable element mariner. Genetics. 1995 May;140(1):183–192. doi: 10.1093/genetics/140.1.183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Matsuzaki H., Nakajima R., Nishiyama J., Araki H., Oshima Y. Chromosome engineering in Saccharomyces cerevisiae by using a site-specific recombination system of a yeast plasmid. J Bacteriol. 1990 Feb;172(2):610–618. doi: 10.1128/jb.172.2.610-618.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McGarry T. J., Lindquist S. The preferential translation of Drosophila hsp70 mRNA requires sequences in the untranslated leader. Cell. 1985 Oct;42(3):903–911. doi: 10.1016/0092-8674(85)90286-7. [DOI] [PubMed] [Google Scholar]
  19. Medhora M. M., MacPeek A. H., Hartl D. L. Excision of the Drosophila transposable element mariner: identification and characterization of the Mos factor. EMBO J. 1988 Jul;7(7):2185–2189. doi: 10.1002/j.1460-2075.1988.tb03057.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. O'Gorman S., Fox D. T., Wahl G. M. Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science. 1991 Mar 15;251(4999):1351–1355. doi: 10.1126/science.1900642. [DOI] [PubMed] [Google Scholar]
  21. Odell J., Caimi P., Sauer B., Russell S. Site-directed recombination in the genome of transgenic tobacco. Mol Gen Genet. 1990 Sep;223(3):369–378. doi: 10.1007/BF00264442. [DOI] [PubMed] [Google Scholar]
  22. Orban P. C., Chui D., Marth J. D. Tissue- and site-specific DNA recombination in transgenic mice. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6861–6865. doi: 10.1073/pnas.89.15.6861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Osborne B. I., Wirtz U., Baker B. A system for insertional mutagenesis and chromosomal rearrangement using the Ds transposon and Cre-lox. Plant J. 1995 Apr;7(4):687–701. doi: 10.1046/j.1365-313x.1995.7040687.x. [DOI] [PubMed] [Google Scholar]
  24. Patton J. S., Gomes X. V., Geyer P. K. Position-independent germline transformation in Drosophila using a cuticle pigmentation gene as a selectable marker. Nucleic Acids Res. 1992 Nov 11;20(21):5859–5860. doi: 10.1093/nar/20.21.5859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pridmore R. D. New and versatile cloning vectors with kanamycin-resistance marker. Gene. 1987;56(2-3):309–312. doi: 10.1016/0378-1119(87)90149-1. [DOI] [PubMed] [Google Scholar]
  26. Rubin G. M., Spradling A. C. Genetic transformation of Drosophila with transposable element vectors. Science. 1982 Oct 22;218(4570):348–353. doi: 10.1126/science.6289436. [DOI] [PubMed] [Google Scholar]
  27. Sauer B. Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jun;7(6):2087–2096. doi: 10.1128/mcb.7.6.2087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sauer B., Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5166–5170. doi: 10.1073/pnas.85.14.5166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sauer B., Henderson N. Targeted insertion of exogenous DNA into the eukaryotic genome by the Cre recombinase. New Biol. 1990 May;2(5):441–449. [PubMed] [Google Scholar]
  30. Spradling A. C., Rubin G. M. Transposition of cloned P elements into Drosophila germ line chromosomes. Science. 1982 Oct 22;218(4570):341–347. doi: 10.1126/science.6289435. [DOI] [PubMed] [Google Scholar]
  31. Van Deursen J., Fornerod M., Van Rees B., Grosveld G. Cre-mediated site-specific translocation between nonhomologous mouse chromosomes. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7376–7380. doi: 10.1073/pnas.92.16.7376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wu C. T. Transvection, nuclear structure, and chromatin proteins. J Cell Biol. 1993 Feb;120(3):587–590. doi: 10.1083/jcb.120.3.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Xu T., Rubin G. M. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development. 1993 Apr;117(4):1223–1237. doi: 10.1242/dev.117.4.1223. [DOI] [PubMed] [Google Scholar]

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